IMPREGNATION
IMPREGNATION
• Also known as INFILTRATION
• Permits the entry of a wax /resin to
provide a form of support needed
during the microtomy process.
• Various waxes and polymers are used
to replace the clearant fount in the
tissue. (Your clearing agents are
replaced by impregnating agents in
order to make one of the goals of
impregnation which is to provide a form
of support needed during the
microtomy process)
• Requires several changes of an
infiltrating agent to displace the clearing
agent (depende kung anong method
gagamitin niyo kung manual or
automated or thru vacuum
impregnation)
• Solidifies the tissue to facilitate
structural rigidity and ease of cutting.
Cellular Morphology
Cellular Structure
*Cell membrane – yung color tan/brown
* This diagram shows junctions between 2
cells, as we all know that cells are not tightly
bound, meron silang gaps in between them,
although they have tight junctions that will help
them adhere with each other if they are to be
processed during the conventional tissue
processes you may observe these spaces
(Extracellular spaces) thru microscopic means
maybe thru electron microscopy. In this
Extracellular spaces dito niyo makikita yung
purpose nung impregnation or infiltration.
Why is Infiltration important?
It solidifies the tissue to a point na hindi siya
sobrang tigas na hindi na mahahati NO! but
rather ang ibibigay lang ng impregnation would
be a type of rigidity that will maintain the shape
of the tissue in order to avoid compression that
may be applied during microtomy process.
This compressive force will be applied as well
to the tissue during the microtomy process
thus it will disrupt the morphological structure
of your cell. Kaya naglalagay tayo infiltrating
agent for us to negate this said compressive
force to not distort the shape of our tissues,
dito papasok yung infiltrating agent.
In the junctions of your cells there is the
presence of Extracellular space that it has not
been filled by your infiltrating agent then
chances are whenever your going to perform
microtomy without the filling of extracellular
spaces then you will observe compressive
forces that may alter the interpretation of the
histopathologist during the tissue examination.
METHODS OF INFILTRATION
1. MANUAL PROCESSING (first and
easiest among the 3, you are going to
introduce your tissues to a molten(
molten ibig sabihin nalusaw na
impregnating agent) impregnating
agent to the tissue in order to allow the
exchange of the clearing agent and the
impregnating agent to replace any fluid
that may be present between the said
junctions that we have so ang
nangyayari dito AGAIN! pinapalitan ng
infiltrating agent yung clearing agent
that may be present in extracellular
spaces thru the use of molten
impregnating agent) (3 changes of your
infiltrating agent is necessary in order to
;1. clear out the clearing agent that may
be present in the site of tissues to make
sure your infiltrating agent has filled the
extracellular spaces of your tissues.)
CERENIO, FERRER, VIDAL
 IMPREGNATION
(matrabaho) (need bantayan kasi a
very hot infiltrating agent can cause
brittleness to your tissues which can
affect the quality or damage the quality
of tissues)
ADVANTAGE: Easily perform and cheap
2. AUTOMATIC PROCESSING (or
automated processing wherein ang
nangyayari dito is that a machine
known a “Tissue processor”;
ADVANTAGES: it will eliminate any
human errors that may have been
present in your manual tissue
processing it is up to you kung gusto
niyong gumaya ng manual tissue
processing or automated tissue
processing kapag nagging
histotechnicians na kayo but most of
the histopathologist uses Automatic
processing para mas mapadali yung
trabaho; it will be quicker compared to
manual processing) (2 CHANGES para
mapalitan yung clearing agent ng
infiltrating agent naten) (it will apply
constant agitation or constant
scheduled agitation thus enhances
further the infiltration process)
(Automated tissue processor)
- From dehydration process until the
end of the infiltration process
- At the end of your tissue processing
will be infiltration
- ADVANTAGE: It could apply constant
scheduled agitation during the
automated process thus enhancing
further the infiltration process. Reduced
human error
- SYNONYM OF AGITATION: swirling
and shaking
3. VACUUM PROCESSING (will apply
vacuum or pressure to 1. Eliminate any
air that may be present in the tissue 2.
To enhance further penetration of your
infiltrating agent inside the tissue
Applying pressure to permit of the
impregnating agent inside the tissues.
(Vacuum could be applied to most
conventional tissue processing
processes). Applying vacuum to
eliminate air present inside the tissue
NOTE!!! VERY HOT IMPREGNATING
AGENT CAN CAUSE BRITLLENESS TO
THE TISSUE
PROPERTIES OF AN INFILTRATING
MEDIUM
➢ Miscible with clearing agent used
(Clearing agents are typically non-polar by
nature is necessary to permit the entry of
infiltrating media)
➢ Must not be too dense (The infiltrating
media must not too dense in order to avoid
hardness that may be encounter during
microtomy process)
o What will happen if the infiltering
media are too dense?
- The tissues may have the
hardness that is similar to bone if the
infiltrating media are too dense thus
it will affect the microtomy process
wherein hindi na makakapag cut ng
maayos and affect the quality tissue
section
➢ Cheap and reliable (the most frequently
used infiltrating media is paraffin because
it is the cheapest among of the infiltrating
media and has a high reliability that can be
used in conventional tissue processing)
➢ Must be compatible with the reagents in
the preceding procedures (The
infiltrating media must be compatible to the
steps after the process of infiltration)
o STEPS AFTER INFILTRATION
- Embedding
- Staining
CERENIO, FERRER, VIDAL
 IMPREGNATION
- Mounting

➢ Formulation must support the structure

of the tissue (the infiltrating medium must
not be too soft that it cannot provide a
rigidity that is needed by the tissue. One of
the goal of infiltration it must provide the
needed rigidity by the tissues, but the
tissues must not be too rigid after infiltration
that cannot be easily cut by the microtome)
o What will happen if the tissue does
not have enough hardness or
rigidity?
- It will easily damage or distort
because of the compress forces
applied during the microtomy
process
➢ Must not contribute opacity to the
tissue; transparent (the infiltrating media
must be transparent in nature. The paraffin
has the transparent appearance that is
necessary for the process of infiltration)
o What will happen if the infiltrating
media or tissue is opaque?
-The image is not clear under
microscope
-The light transmitted are fewer
compared to when tissues or
infiltrating media is transparent

CERENIO, FERRER, VIDAL

 IMPREGNATION
IMPREGNATION
• Also known as INFILTRATION
• Permits the entry of a wax /resin to
provide a form of support needed during
the microtomy process.
• Various waxes and polymers are used to
replace the clearant fount in the tissue.
(Your clearing agents are replaced by
impregnating agents to provide a form of
support needed during the microtomy
process)
• Requires several changes of an
infiltrating agent to displace the clearing
agent (depende kung anong method
gagamitin niyo kung manual or
automated or thru vacuum impregnation;
3 to 4 changes)
• Infiltrating agents should be soluble with
clearing agents
• Solidifies the tissue to
facilitate structural rigidity and ease of
cutting.
Cellular Structure
* This diagram shows junctions between 2 cells
and they have gaps in between them, during the
conventional tissue processes you may
observe these spaces (Extracellular spaces)
thru microscopic means maybe thru electron
microscopy.
* If clearing agents is not remove in the spaces
it will look like a sponge and parang may water
na lalabas during microtomy
Why is Infiltration important?
- It solidifies the tissue
- Provide rigidity that will maintain the shape
of the tissue in order to avoid compression
that may be applied during microtomy
process.
- Compressive force will be applied during
the microtomy process can disrupt the
morphological structure of your cell
METHODS OF INFILTRATION
1. MANUAL PROCESSING
- Easiest
- You are going to introduce your tissues
to a molten (molten ibig sabihin nalusaw
na impregnating agent) impregnating
agent to the tissue in order to allow the
exchange of the clearing agent and the
impregnating agent to replace any fluid
that may be present between the said
junctions
- We can monitor the completeness of
infiltration
- 3 changes of infiltrating agent is needed
- Tidious
- Time consuming
- Very hot infiltrating agent can cause
brittleness to your tissues which can
affect the quality or damage the quality
of tissues
- Cheap
- Prone to human error
2. AUTOMATIC PROCESSING
- We use a machine known as “
Automated Tissue Processor” which
perform dehydration, clearing and
infiltration for you
- ADVANTAGES:
o it will eliminate any human errors
o it will be quicker compared to
manual processing
o 2 CHANGES is needed
o it will apply constant agitation or
constant scheduled agitation thus
enhances further the infiltration
process
- DISADVANTAGE
o Not cost effective
CERENIO, FERRER, VIDAL
 IMPREGNATION
SYNONYM OF AGITATION: swirling and
shaking
3. VACUUM PROCESSING
- Application of vacuum or pressure to:
o Eliminate any air that may be
present in the tissue
o To enhance further penetration
of your infiltrating agent inside
the tissue
- Quicker than manual and automatic
processing
NOTE!!! VERY HOT IMPREGNATING
AGENT CAN CAUSE BRITLLENESS TO
THE TISSUE
PROPERTIES OF AN INFILTRATING
MEDIUM
 Miscible with clearing agent used
- Clearing agents are typically non-polar
by nature is necessary to permit the
entry of infiltrating media
 Must not be too dense that can
contribute to excessive hardness of
tissues
- If the infiltrating media is to dense the
tissues may have a hardness that is
similar to the bone if the infiltrating
media are too dense thus it will affect
the microtomy process
 Cheap, reproducible and reliable
- the most frequently used infiltrating
media is paraffin because it is the
cheapest among of the infiltrating media
and highest reproducibility/reliability
rate
 Must be compatible with the reagents in
the preceding procedures
STEPS AFTER INFILTRATION
- embedding
- microtomy
- Staining
- Mounting
 Formulation must support the structure
of the tissue
- the infiltrating medium must not be too
soft that it cannot provide a rigidity that is
needed by the tissue.
- Infiltrating agent should be easily solidify
at room temperature in order to provide
the strength of the tissue
- Infiltrating medium should be liquid in
nature and loss its viscosity when it is
subjected to heat and when it cools down
it increases solidity
- Molten nature of the infiltrating medium
is necessary for it to enter the
extracellular spaces of the cell
 Must not contribute opacity to the tissue;
transparent
- the infiltrating media must be transparent
in nature so that it will not affect the
image of the tissue. The paraffin has the
transparent appearance that is
necessary for the process of infiltration
CERENIO, FERRER, VIDAL
PARAFFIN
- Simplest, most commonly used
infiltrating agent.
- Also considered as the best because of
high reproducibility rate and high
reliability
- Mixtures of purified wax (paraffin) and
various additives (e.g., plastic resins,
antioxidants, dyes, other additives, and
hydrocarbons will contribute to the
quality of paraffin wax, and it is
necessary to meet the certain
characteristics that we need during the
melting the of paraffin wax.
- Paraffin can be used on its own so that
it will not hinder or stop any processes
so that it will give better results during
conventional tissue processing
- Solid at room temperature
- Melting point (MP): approximately 60
degrees Celsius, melts faster at 65-70
degrees Celsius
- Most commonly used for routine
histopathology has an MP of 56-58
degree Celsius
- The density of paraffin wax can
contribute to the melting point of
paraffin wax. The more paraffin inside
the wax, the lower the melting point of
paraffin wax will become, and it is very
important of melting the wax during
infiltrating process
ALTERNATIVE INFILTRATION MEDIA:
➢ An alternative to paraffin is necessary
when these are observed
o Processing will remove several
components of the tissue that is for
investigation (for example lipids)
o Using heat may detrimentally
affect several components of the
cell (for example lipids and
enzymes)
o Thin sections are necessary (i.e.,
Electron Microscopy.) the thinnest
of section is very important and this
is impossible if the tissues is
infiltrated and embedded in a
paraffin media
o The infiltrating medium cannot
support the tissue as it is not hard
(double embedding: is the process
by which tissues are first
embedded or fully infiltrated with a
supporting medium such as agar
or nitrocellulose then infiltrated is
second time with paraffin wax and
this is necessary when you
process certain tissues that have
been embedded by other media.
Paraffin is still essential for
infiltrating process when
performing double embedding but
there are some qualities of
embedding media that can
contribute to the quality of the
tissue thus paraffin can be avoided
by now or it can be first embedded
or infiltrated using alternatives to
paraffin like for example GELATIN
OR AGAR
SUBSTITUTES COME IN THE FORM
OF:
o Resins and other waxes
o Celloidin
o Agar
o Gelatin
o Plastics
Can come in one or in combination
with the other as the process of
double embedding
ALTERNATIVE INFILTRATION MEDIA:
RESINS
Used for embedding and infiltrating
tissues for special procedures (e.g.,
Electron microscopy, ultra-thin
sectioning for high resolution studies,
and use resins in undecalcified bone,
Immunohistochemistry IHC)
Use in conventional tissue processing
instead of using electron microscopy
we can use light microscopy
Provides exceptionally strong
support to tissues harder compared
to the paraffin wax
(Ex. Electron microscopy since the
tissues are being bombarded with
electrons during the process of electron
microscopy)
CERENIO, FERRER, VIDAL
 Allows for a thinner sectioning vs.
paraffin (paraffin section can often
come in section with 4-6 micrometers
but in electron microscopy we are using
section that ate thinner than 4-6
micrometer in thickness thus resin is
very much advisable when it comes to
infiltration and embedding of tissue
sections for electron microscopy
Examples of currently available resins
include epoxy, acrylic, and polyester
formulations, either pure or mixture of
these (but typically the ones that are
use is puriform of these formulations)
Polyester Resins- first used among
the three (since 1950`s)
Epoxy plastics: Bisphenol A (Araldite),
Glycerol (Epon), or Cyclohexane
dioxide (Spurr)
Acrylic Plastics: Glycol methacrylate
(GMA) and Methyl Methacrylate (MMA)
PLASTIC (RESIN) EMBEDDING
➢ Used particularly in hard tissues such as
undecalcified bone and for high resolution
light microscopy of tissue sections thinner
than the usual 4-6 µm, such as renal
biopsies and bone marrow biopsies.
➢ Plastics are classified into epoxy,
polyester, or acrylic, based on their
chemical composition.
o Epoxy embedding plastics are made
up of a carefully balanced mixture of
epoxy plastic, catalysts, and
accelerators.
o Three types of epoxy plastics are
used in microscopy, i.e., those
based on either bisphenol
▪ A (Araldite), or glycerol (Epon),
or cyclohexene dioxide (Spurr).
▪ Infiltration by Araldite is slow,
partly because the epoxy plastic
itself is a large molecule.
▪ The glycerol-based epoxy
plastics (Epon) have a lower
viscosity but are often sold as
mixtures of isomers.
▪ Cyclohexene dioxide-based
plastics (Spurr) can be
obtained pure, have very low
viscosity, and infiltrate fastest.
SPURR’S RESIN
- Is a Low Viscosity mixture which provides
rapid infiltration of tissues. It's easy to
prepare and mixes rapidly.
- This resin is compatible with ethanol so no
change to propylene oxide is needed prior
to infiltration.
- Polymerization at 60°C is
recommended.
DISADVANTAGES OF EPOXY
PLASTICS
- They are hydrophobic and subsequent
oxidation by peroxide to correct this may
produce tissue damage.
- Epoxide groups may reduce antigenicity of
embedded tissue and may compromise the
result of immunohistochemical staining.
- More importantly, epoxy resins may cause
sensitization if absorbed by skin or
inhalation.
The components of many epoxy plastics
are toxic and one of its components, vinyl
cyclohexane dioxide (VCD) is known to
be carcinogenic.
Polyester plastics were originally
introduced for electron microscopy in the
mid- 1950s, but have been superseded by
more superior epoxides, and are now
seldom used.
Acrylic plastics are made up of esters of
acrylic or methacrylic acid, and are used
extensively for light microscopy.
The polar water soluble, 2-hydroxyethyl
methacrylate, commonly known as "glycol
methacrylate", or GMA,
ALTERNATIVE INFILTRATION
MEDIA: OTHER WAXES
PARAPLAST
- Mix of highly purified paraffin and synthetic
plastic polymers
- Melting Point: 56-57 degree Celsius
CERENIO, FERRER, VIDAL
- More elastic and resilient than paraffin thus
it can reduce the distortion of tissue during
the microtomy process as it will revert back
to its original shape once the compression
during the cutting has finished
- Advisable for large dense tissue blocks
(e.g., bones, brains)
EMBEDDOL
- Synthetic wax substitutes similar to
Paraplast
- Melting point: 56-58 degree Celsius
- Examples:
o Bio/aid- recommended for
embedding eyes
o Tissue Mat- paraffin with rubber;
same property as that of paraplast
ESTER WAX
• Harder than paraffin
• (because of its density) Has a lower MP
vs. paraffin (46°C - 48°C)
• Soluble with organic solvents (i.e., 95%
ethanol, clearing agents)
• Indicated if cellosolve or xylene is used
as a clearing agent.
• May be used as an infiltrating agent if
CLEARING will be emitted.
WATER SOLUBLE WAXES
• Plastic polymers (PEG) – ALTERNATIVE
Polyethylene glycol
• MP: 38°C - 42°C or 45°C- 56°C
• Blended with histological paraffin wax
to improve adhesion, hardness, and
plasticity of the agent.
CARBOWAX
• PEG water- soluble wax
• May be used if dehydration and clearing
will be omitted suitable for infiltrating
tissues for enzyme histochemistry
(enzymes can be deteriorated easily by
heat thus, u can preserve enzymes by
using carbowax) studies since tissues
will not be subjected to too much heat.
• Highly hygroscopic
ALTERNATIVE INFILTRATION MEDIA:
CELLOIDIN
• A.K.A. COLLODION
• Purified nitrocellulose
• Suitable for specimens with large,
hollow cavities (such as intestines);
dense tissues (such as bones); embryo;
soft, delicate tissue sections of mixed
consistency.(such as the brain)
• Comes as a thin, medium, or thick
solutions of cellulose dissolved in equal
parts of ether and alcohol.
There are 2 types of embedding using
the celloidin method
1. Wet Celloidin embedding method
2. Dry Celloidin embedding method
NITROCELLULOSE/ LOW VISCOSITY
NITROCELLULOSE
• Another type of celloidin soluble in an
ether-alcohol solution
• Less viscous
• Allows for a thinner sectioning
• Plasticizers (e.g., castor oil) (extracted
from the plant Oleum ricini) may be
added to the solution to reduce the
tendencies of tissue cracking
INFILTRATION MEDIA:
GELATIN
Gelatin can be used as double-
embedding medium
• Primarily used in the production of
whole organ sections with using a
mod’d. Gough-Wentworth technique
• Rarely used; dehydration must be
avoided
• Generally used as an embedding agent
for frozen sections and for delicate
specimens.
First, infiltrate the tissues with gelatin
and then infiltrate using paraffin
ALTERNATIVE INFILTRATION MEDIA:
AGAR
• Does not provide enough support
during sectioning.
CERENIO, FERRER, VIDAL
 • Used on small, friable tissue pieces
after fixation to provide additional
structure (double-embedding)
When performing double embedding:
Perform infiltration immediately after
the fixation process then proceed with
other processes then during the
infiltration process u are going to
infiltrate the tissue using paraffin
CONSIDERATIONS DURING
IMPREGNATION
GENERAL:
• The temperature must be closely
monitored (lipids are heat labile)
• Several changes of the infiltrating agent
is necessary to remove the clearing
agent
In manual infiltration processing this
will now require you to perform 3-4
changes of impregnation or
impregnating media
For automated process, this will only
require you to change to 3 times more
or less typically ranges 2-3 is enough
to infiltrate completely.
• The shorter the time of infiltrating the
tissues, the better (to avoid brittleness)
Typically, 15 minutes to finish for a
single change
• Whatever agent is used in the
infiltration process, the same material is
used in the embedding process.
• A homogenous agent is necessary. (will
produce crystals that are very much
compatible)
PARAFFIN:
• The temperature set to melt the wax
must be at least 2°C - 5°C above its
melting point (oven will be set at 58°C
to melt paraffin wax)
A molten state of paraffin wax is
necessary for impregnation process to
proceed, if paraffin wax is not molten
then clearing agent will not be removed
from the tissues. This molten state is
very much necessary to the entirety of
paraffin wax medium.
• Paraffin wax with an MP of 56°C is
routinely used
• The ambient temperature will dictate
what wax with a preset MP will be used:
o RT at 20°C- 24°C = Wax with
54°C – 58 °C MP
o RT at 15°C – 18 °C = Wax with
50 °C- 54°C MP
• Pure
• Fresh wax must be filtered before use
• Trimmed wax can be reused (galing sa
excess)
• A special filter paper is necessary to
filter the wax (i.e., Green’s No. 904)
Can only be used twice becoz of high-
water content after it has been reused.
If u want to eliminate the water present
in paraffin wax, heat the paraffin wax
100- 105°C to boil off any excess water
molecules
OTHER INFILTRATING MEDIA:
• Schedules for changing varies per
infiltrating media. (e.g., Celloidin
method will take days to infiltrate)
• MP temperature varies per infiltrating
media.
• Follow manufacturer’s
recommendation when using media.
• Some infiltrating medium have
advantages, disadvantages, and
indications for use (e.g., gelatin for
frozen sections) (resins for electron
microscopy)
CERENIO, FERRER, VIDAL
 MICROTOMY
MICROTOMY
➢ Greek word: (mikros: small), (temnein: to
cut) (Temnien refers to the cutting of
embedded tissues)
➢ Process of uniformly cutting embedded
tissue specimens into thin slices (sections),
forming RIBBONS (thin samples are put in
the top of the glass slide and placing the
sections on the glass slide and proceed to
the de-paraffinization process and staining
which will ultimately provide sample
needed for examination)
- RIBBONS: this will allow you to place
multiple tissue sections on a single glass
slide. The quality of ribbons will depend
on tissue processing
➢ Allows study of tissues at a microscopic
level (the tissue sections are cut in very thin
slices around 2-4 micrometers or even
thinner and it is necessary so that the light
can pass through the tissue section which
is needed for the microscopy process.
Because if the tissue section is very thick
they cannot pass through to the tissue
section)
➢ The process of microtomy will also apply to
the process of frozen tissue sectioning
THE MICROTOME
➢ Device that cuts tissue blocks into thin
sections
➢ Consists of these three main parts:
- Block Holder also known as CHUCK:
This will hold the tissue blocks in place
during the process of forming thin
sections.
- Knife Carrier and Knife (AKA cutting
parts): this will hold or carry the knife in
place during the microtomy process
- Pawl, Ratchet Feed Wheel, and
Adjustments Screws
(Mechanical/Moving parts)
o PAWL AND RATCHET FEED
WHEEL are mechanisms/
mechanical parts that are moving
inside the microtome
o ADJUSTMENT SCREWS: Adjust
the different measurements of the
microtome process through
cutting.
- In modern microtomes they are
automated or semi-automated and they
already program if they will be adjusted
or not.
- In manual microtomes, we are the ones
to adjust the tissue block holders using
adjustment screws manually
TYPES OF MICROTOMES
1. ROTARY (MINOT)
- Invented by Minot (1885-1886)
- Most commonly used in clinical and
research laboratories
- Actual cutting is based on the rotary
action, cranking the flywheel (in the
microtomy process it will depend on the
skill of histotechnician and also to the
force applied by rotating the flywheel
▪ HOW DO YOU OPERATE THE
ROTARY MICROTOME? By
simple rotating the flywheel
- Knife is fixed. The tissue block holder
moves down for cutting and advancing,
and up for repositioning
o In the down stroke of tissue
block will allow you to cut tissue
sections and to move forward the
tissue block holder to cut the
tissue sections
o In the upstroke, it will reposition
and ready the tissue blocks for
cutting
o Half of the revolution of flywheel
consist of the down stroke and
the other half will consist of up
stroke of tissue block holder
- Can cut sections with 2-4 micrometers
thickness (Bancroft)
▪ Bruce Gregorios: 3-5
micrometers
▪ Dey: 2-3 micrometers
- Advisable for paraffin and celloidin
embedded tissues
CERENIO, FERRER, VIDAL
 MICROTOMY
2. SLIDING (ADAMS)
- Invented by Adams (1789) and
considered as oldest microtome and
dangerous type of microtome because
the knife is being manipulated and not
the tissue block holder and the knife is
exposed where in there is a parts na
hindi natatakpan ng mga knife carriers
- Ideal for cutting sections with thickness
greater than 3um
- Knife can be set flat for paraffin-
embedded tissues (soft tissues); oblique
(diagonal) for celloidin-embedded
tissues (hard tissues)
- Most dangerous type; exposed moving
blade
- The block or the blade that is slided
forward or backward creating the sliding
motion
2 TYPES OF SLIDING ADAMS
A. STANDARD SLIDING
o Specimen slides under the blade
while cutting OR the block
remains stationary (buttom) while
knife glides over
o Good for celloidin-embedded
tissues
o We can set the greater angle of
the knife compared to the rotary
microtome and used to cut the
celloidin embedded tissues
B. BASE-SLEDGE
o Slid is being pushed to create
tissue sections
o Consists of 2 pillars which holds
the knife; specimen passes
below the blade (The knife is kept
in place and slid is the one that is
being move)
o Ideal for sectioning tough or large
blocks with >10 um thickness
o Originally designed for sectioning
very large blocks and whole
organs like whole brains sections
and specimen that is embedded
with embedding medium
o Heavier; more stable (the
heaviness of the microtome will
have an advantage during
microtomy process. The heavier
the microtomes are the more
stable it will be)
3. ROCKING (CAMBRIDGE)
o invented by Paldwell Trefall (1881)
o simplest
o lever-action
o sections are cut in a slightly curved
plane, with 10-12 um thickness
o available in two sizes that allow you
to cut small and large paraffin blocks
o operated by cranking the lever
(push down to cut down tissue)
o cutting section through rocking
motion thus making tissue sections
that are uneven
o because of the observable creation
of an arch during microtomy you can
create uneven sections
4. FREEZING (QUECKETT)
o Invented by Queckett (1848)
o Block holder is circular shaped,
hollow, and perforated and is attached
to reinforced flexible lead pipe
o Lever allows for quick, intermittent flow
or burst of cryogenic agent
o Injected by cryogenic agent to maintain
the frozen state of tissue section and
maintain the cold temperature of the
knife
o A second cooling mechanism
maintains the cold temperature of the
knife
CERENIO, FERRER, VIDAL
 MICROTOMY
o For rapid diagnosis; for frozen tissue
section
o Tissue samples are placed on top of
tissue block holder and will burst of
cryogenic agent that is being delivered
through a reinforced lead pipe. With
the operation of lever, you will apply
short intermittent burst of the cryogenic
agent to freeze ad to maintain frozen
state of tissue section. Manipulation of
other lever, you can maintain
temperature of knife (balikan yung
temperature ng knife using freezing
microtome)
o Cryogenic agents for freezing
microtome
▪ Liquid nitrogen
▪ Isopentane cooled with
liquid nitrogen
▪ CO2 in liquid or gas
▪ Acetone
o Cuts undehydrated thin to semi-thin
sections
o Fresh or frozen tissues
o Ideal for fat (mesenteric specimens
found at the lining of intestine, breast
specimen) and neurological specimen
(myelin sheath) cutting, also for tissues
easily destroyed by heat
o Same mechanism as that of the sliding
microtome by manipulating the knife
using sliding motion
Note!!! Alternative for freezing microtome
is cryostat or cold microtome
Cryostat (Cold microtome)
- Consist of a refrigerated chamber that
freezes tissues to a correct degree of
hardness’
- Contains a rotary rocking microtome
(operated similar to rotary microtome
and cut tissue similar to rocking
microtome)
- Chamber is maintained at a temperature
of -5°C- -30°C (average- 20°C)
- Provides tissue sections with 4 um
thickness
- Ideal for preparing thin sections of fresh
frozen tissue section for:
▪ Rapid diagnosis
▪ Fluorescent antibody staining
▪ Enzyme histochemistry
o Produces 1 section at a time like
freezing microtome because we
removed EMBEDDING
o PRESS – placed on top of tissue
section so that cooling/solidification
process of frozen tissue section will be
hasten and will keep tissue at uniform
and flat shape
o Equipment use in cryostat is maintain at
a temperature used in chamber itself
(glass slide,brush) kept at -20° C inside
the chamber
o Above the knife we can see tissue
sections and we need to brush it of in
order for it to be placed in glass slide
and not to destroy the quality of tissue.
You can use finger moistened with
water to pick up tissue sections.
Because of the use of water you can
form ice crystals or freezing artifacts
5. UNTRATHIN MICROTOME
o Electron microscopy
o Uses a knife made of glass or gem-
graded diamaond knife to cut sections
with 60-100 nm thickness (Dey 40-100
nm)
o Semi-thin sections with 0.5-1 um thick
can also be produced when a glass
knife or an industrial-grade diamond
knife is equipped
o Tissue blocks for electron microscopy
that is subjected to ultrathin microtomy
is kept at a cube shape side that 1 mm
MICROTOME KNIVES AND BLADES
- Knives of different shapes, sizes and
material allow for the cutting of different
degrees of tissue and embedding media
hardness
- Disposable blades are already available
for use
- Outdated
- Since we need to maintain sharpness of
knives we can use disposal blades
MICROTOME KNIVES
- Can be made of high grade steel (made
of tungsten carbide or steel that has high
carbon content), glass, or diamond
especially those that will be used for
electron microscopy
- Knives should have high carbon content
para hindi agad maging mapurol
CERENIO, FERRER, VIDAL
 MICROTOMY
- Provides thin secretions (ave. 2 um)
- Knife edge must be maintained to
provide thin sections
- Different profiles, different uses
(thickness of tissue section or
denseness of embedding media)
- Downside of microtome knives: after
every shift kailangan mahasa yung mga
knives to maintain sharpness
KNIFE PROFILES
- Profile A (Planoconcave/Planeconcave)
o 25 mm length
o For cutting celloidin and paraffin
tissue
o Used on both sliding and base
sledge, rotary, or rocking microtomes
o Less concave edge for celloidin-
embedded blocks
o More concave side for paraffin-
embedded blocks; use for tissue that
is not that dense like soft tissue
blocks

- Profile B (Biconcave)
o 120 mm length
o Recommended for cutting paraffin
sections on rotary microtomes
o Both sides are kept at concave shape

- Profile C (Plane-Wedge/Wedge)
o 100 mm length
o Frozen sections
o Extremely hard and tough specimen
on paraffin (using base sledge)
o Standard profile out of all microtome
knives
o For routine purposes
o Advantage: has very high carbon or
very stable shape content

- Profile D (Tool Edge/Chisel)

o Plain sides with a steep edge
o For cutting very dense tissues
(undecalcified bone)
o The chisel tip of the knife makes it
hard to hone

CERENIO, FERRER, VIDAL

 MICROTOMY PART II
DISPOSABLE KNIVES AND BLADES
- More commonly used nowadays since
knives and blades are easy to replaced
- An adapter can be attached to knives to
hold these blades
- Dummy knife: type of knife that is
equipped in a microtome which has a dull
edge; mapurol na knife and its main
purpose is to placed adapter so that you
can equipped disposable knife
- Two profiles:
o Low-profile: soft tissues, large but
soft tissues; has lower carbon
content; thinner
o High profile: firm and relatively
hard tissues (decalcified
bone/muscle)
GLASS AND DIAMOND KNIVES
- Incorporated in ultrathin microtomes
- Produces the thinnest type of sections (40-
100 nm in ultrathin)
- Glass
o Cheap; easily replaced
o For very hard tissues
- Diamond
o Expensive; relatively hard to
sharpen
o For epoxy resin-embedded tissues
OTHER TOOLS AND EQUIPMENT
WATER BATH (FLOTATION CHAMBER)
- For floating tissue sections, after cutting
- Set warm temperature, often controlled by
a thermostat
- Used for tissue sections that form tissue
ribbons; only used in embedded samples
not for frozen tissues
- Temperature must be at least 5-10C below
the MP of the wax;
- Preferably, distilled water must be used to
avoid bubble formation
o Tissues sections should be bubble
free so that tissues sections will
adhere to the glass slides
- Adding alcohol or a small drop of
detergent/soap helps in reducing surface
tension
- Used to flatten/straighten out tissue
sections to reduce surface tension
BRUSH, TEASING NEEDLED AND
FORCEPS
- Removes creases, folds, and bubbles in
the section ribbons
- Can also be used in manipulating the
ribbon during the microtomy
- Brush: removes sections off of the blade
or any excess paraffin that clung to the
microtome knife and can be used for
cleaning the microtome
- Use to transfer tissue sections into water
bath
Xylene- used to clean microtome; brush first
then apply xylene to areas that are exposed to
paraffin
SLIDES, PENCILS AND PENS
- Glass slides: dimension 76X25 mm, with
thickness of 1.0-1.2 mm;
- Larger slides may be used for larger
specimens like brain
- May have a frosted side to which can be
used to etching or writing the ID of the
patient
- Adhesive must be applied on the side
- Pens and pencils: must be chemical
resistant which can’t be affected by
reagents used for mounting and staining
- Diamond tip pencil: used for
etching/inscribing information in glass
slide
ADHESIVE
- Prevents tissues from detaching from
the slide
- Comes in different formulation
o Protein adhesive (like Mayer’s
Egg Albumin, dried albumin)
o Gelatin (1%): starch
o Gelatin-formaldehyde mixture
o Poly-L-lysine- for IHC
o 3-aminopropyltriethoxysilane
(APES)- for bloody specimens;
cytological studies
o Human plasma is can be used for
adhesives for whole blood
samples. However, it can affect
staining
- Some laboratories may also use slides with
a permanent positive charge (coated
with a basic polymer that will react with the
CERENIO, FERRER, VIDAL
 MICROTOMY PART II
amino component of proteins that can
be found in tissue samples that create
covalent bond that act as adhesive)
OTHER TOOLS AND EQUIPMENT
DRYING OVEN/HOT PLATE
- It is used to melt out the paraffin or any
wax that have been used as embedding
and infiltrating medium
- This will enhance further the adhesion
of tissues in glass slide
- Before staining process, we must de-
paraffinize the slide by either using
xylene to remove the paraffin or place
tissue section on a drying oven or hot
plate
- Design with a fan inside the oven to
circulate the hot heat inside the oven
- Must be set approximately to that of the
melting point of the wax used
- The drying process will take you 30
minutes for the regular tissue process
- For drying slides and
deparaffinization process
- For delicate specimens; it`s better to
use lower temperature (37 degree
Celsius for 24 hours)
KNIFE SHARPENING
- Maintains the razor-sharp edge of
microtome knives
- Repairs badly-nicked knives and dull,
blunt edges
- HONING (Hard sharpening)
▪ Removal of gross nicks on the knife
edge (Course) and also to grind the
edge to create an even edge (if you
cut dense tissue pwedeng
matapyasan yung edge ng mga
microtome knives yun yung
tinatawag na nick. Thus, this nick
must be remove para hindi
magkaroon ng uneven tissue
section
▪ Grinds the edge to create an even
edge (Honing proper)
- STROPPING
▪ Removes burst that form during the
sharpening process
▪ Polishes the knife
▪ Otherwise known as the knife
polishing process
HONING
- For sharpening
- “Repairs” the knife and maintain the
knife edge
- Make use of a honing stone
(whetstone) to sharpens the knife
- Degree of sharpness= finesse/grit of
the abrasive used
- The finer grit (smooth) it can produce
sharper knife but coarser they are used
for repairing badly nicked knives like
carborundum
HONING STONES
1. FINE CARBORUNDUM
for - Coarsest of all the whetstone (low grit)-
magaspang
- For repairing badly nicked knives,
followed by honing with a Belgian
yellow stone or Arkansas stone
- Can also be used for coarse honing
- During the process of honing, start with
a lower grit and going to a stone with a
higher grit (mas magaspang papunta
sa mikinis na bato)
2. BELGIAN YELLOW/ BELGIUM
COTICULE
- Less coarse vs. carborundum stone
- Provides a better polish after honing
- If you used the smoother side which
has finer grit or higher grit, then it has
the best results for honing
- Used for blunt or nicked edges
- It has two sides: one it has abrasive
side compared to other side
- For coarse honing effect, carborundum
is better than the Belgian yellow
CERENIO, FERRER, VIDAL
 MICROTOMY PART II
3. ARKANSAS
- Provides a better finish out of all the
stones
- Better polish vs Belgian yellow because
of its smoother surface
- Best to used in microtome knives
- It has a very smooth surface that has a
highest grit of all stones
- The degree of sharpness will depend
on the finest or smoothness of honing
stone
- The smoother the honing the stone is
better to make your knives sharper
- The kitchen knives is the same process
of honing the microtome knives
STROPPING
- Running the edge of the blade against
a leather surface (strop).
- Strop ca be made up of quality horse
leather which will place in puddle, and
we call it as PADDLE STROKE
- Polishes the knife
- Removes burst that may have formed
during the honing process
- BURST: fine sharp – that can be found
on the edge of knife during honing
process and can only observed by a
trained eye
- It is necessary to polish the knives after
the honing process in order to create a
good quality tissue section.
HONING PROCESS
1. Surface of the stone is cleaned with a
soft cloth moistened with xylene to
remove any impurities or any
coarseness that may formed previous
during the honing process
2. A thin layer of clove and mineral oil,
xylene, liquid paraffin, or soapy water is
applied on the stone, and this will form
a thin film of lubrication during the
honing process. In honing the knife
there is fine sand that is formed out of
friction/abrasion formed by the knife
and honing stone. So, this abrasion can
form irregularities during the honing
process.
3. The heel (handle end) is drawn
diagonally towards the operator until
the toe (head portion) reaches the
operator
4. The knife is flipped and is dragged in
the similar manner as that of the other
edge; EDGE FIRST, in a HEEL to TOE
fashion ( we need 20-30 double strokes
on each sides)
STROPPING PROCESS
1. Use paddle stroke or leather belt that
has been moistened with oil. The knife
is laid almost flat on the strop
paddle/leather strop, at the top end.
2. With a toe to heel pattern, the knife is
pulled towards the user
3. The knife is flipped, and the toe to heel
pattern of dragging is repeated on the
other side
4. 40-120 double strokes are done on
each side to polish the knives
SECTIONING
THERE ARE THREE GENERAL
TYPES OF SECTIONS:
o Paraffin sections- The most
common among all of the types of
sections
o Celloidin sections- used in whole
organs
CERENIO, FERRER, VIDAL
 MICROTOMY PART II

o Frozen sections- consist of fresh - 5-100 C to prevent uneven sections and

tissue that have been frozen using a
cryogenic agent like carbon dioxide
gas, liquid nitrogen, isopentane
cooled liquid nitrogen and acetone.
Frozen section is cut using a
freezing or cold microtome and for
celloidin and paraffin are cut either a
rotary, rocking, or sliding microtome
depending on denseness of tissue
block
used in routine purposes/optimal angle
- Minimal angle will be 00 C and
maximum will be 150 C
- 150 C allows better penetration to the
tissue, minimizing sections
- Lower angle= less=friction= less
compression
- Less compression is necessary to avoid
the damage of tissue sample or section

TRIMMING
- This will allow you to fit the tissue block
inside the tissue block holder and
exposed the tissue sample within the
tissue block
- The sides, top and bottom are trimmed
until it is level, and the sides must be
parallel on each of the sides of tissue
block
- Done using an old, but sharp knife
- COARSE FACING/ COARSE
TRIMMING may be done to expose the
tissue sample, and this is done at 30
micrometer intervals. Coarse facing
must be done carefully to prevent the
damage of tissue blocks.
- FINE TRIMMING makes the face tissue
block smoother, for creating better
tissue sections. The knife is elevated at
a clearance angle around 0 -15 degrees

ANGLE OF MICROTOMES

SUGGESTS POSITIONING TO
ALLOW BETTER SECTIONS

1. RAKE ANGLE
- Angle between the bevel of the knife
and the imaginary perpendicular line
from the surface of the block
2. CUTTING ANGLE/ BEVEL ANGLE
- Angle formed from the cutting edges of
the knife 27–32-degree Celsius
3. CLEARANCE ANGLE
- From the surface of the block to the
cutting facet (bevel)

CERENIO, FERRER, VIDAL

 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES: STAINING
STAINING
- After creating tissue sections, samples
must be stained to create nice
histological slide
- Use to distinguish different cellular
structure, tissue and cellular
morphologies
- Process of introducing an unstained
specimen to a dye/stain
- Fine structures of the cell are
demonstrated with the use of stains;
helps the visualization of different cell
components or other extracellular matrix
(every part of samples imparts color)
- Stains add contrast to the different
structures of tissues, depending on their
affinity to the stain and the intensity of
the stain (most staining procedures will
use primary stain and counter stain
which will provide contrast)
- Gram stain: crystal violet and safranin
- Acid fast stain: carbol fuchsin
- Methylene blue: stain structures that will
take up methylene dye like nucleus and
basophilic granules
- Eosinophil: stain the cytoplasm
Contrast
- Stains will work separately to distinguish
different structures
Note!!!
- The principle of staining will work for
most sample in the laboratory whether it
is histological slide, peripheral blood
smear or bone marrow aspirate.
- All specimens that you have created a
slide are considered as histological
slides
PRINCIPLE OF STAINING
- Different cell components are
transparent and colorless
- The extracellular matrix and different
cellular components pick up these stains
base on their affinity to the stain
o There are some parts that are
attracted to basic dyes and some
are attracted to acidic dyes
o Acidic dye: attracted to
structures that are acidophilic
because the structures are basic
o Basic dye: attracted to structures
that are basophilic because the
structures are acidic
- Stains form COLULOMBRIC
REACTIONS or SALT BRIDGES (for
the stains to retain even if they were
decolorized) that arise from the electrical
attraction of the stain with the cell
components
o Hematoxylin: because of its
basic property it will be attracted
to the nucleus which is acidic
o Crystal violet: attracted to the
cell wall and with the help of
mordant the stain will remain in
the structure and will form a salt
bridge
- Some tissue samples must be
decolorized to discern different
structures
CELLS ENHANCE THE VISUALIZATION
AND EXAMINATION OF THE PARTS OF
INTEREST IN TISSUE SAMPLES AFTER
STAINING:
 Metabolic process
 Differentiating live from dead cells is
necessary for sperm analysis
 Demonstrate the relationship between
internal and external cellular structures
 Identify different types of cells like in
grams staining and peripheral blood
smear examination
 In hematology: stained blood
smear
 In bacteriology: stained bacterial
slides
 In histopathology: staining
property of tissue sample
STAINS
 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES: STAINING
- The color of the dye is not the real color - Mordants are integral to the procedures;
of the tissue (the unstained slide is the
original color of the tissue but rather the
tissue is demonstrating the color of the
dye that has been used)
- Most of the histological procedures make
use of the hematoxylin and eosin
staining process.
- The routine H&E staining procedure
because it is
o Quick
o Cheap
o Informative
CYTOPLASMIC: stains by eosin
NUCLEAR MATERIAL: stain by
hematoxylin
HISTOLOGICAL STAINING
- Tissue components and the general
relationship of cells or tissues are
visualized through direct interaction with
a dye or staining solution.
- All types of staining procedures fall into
the category of histological staining
procedures as long as you have sample
from the patient and stained that is the
example of the histological slide, and
whenever you stained that, example of
histological staining
METHODS OF STAINING
DIRECT STAINING
- Also known as SIMPLE STAINING
- Only one type of dye is used, and tissue
will take up the dye that will provide a
color
- A single color is seen after the process
- The dye stains one component that has
an affinity to the dye
- Ex. Malassezia furfur is more
distinguishable with the use of
fluorescence microscopy
INDIRECT STAINING
- It stains the background
- Also known as NEGATIVE STAINING
because it does not stain the cells but
rather the background
acts as the bridge between the tissue
and the stain
- Mordant binds with stains to form a
colored “lake” combining with the tissue
- Insoluble “tissue-mordant-type” complex
- Allows counterstaining procedures
- The dead cells stained red by the eosin
and live sperm cells will not pick up the
stained because they have a resistance
of stains used
- INDIA INK: stains the background with
the color brown or black and the fungus
(Cryptococcus neoformans) will not take
up the stains
PROGRESSIVE STAINING
- Increasing the intensity of the color by
soaking the tissues until you acquired
the desired color
- Ex. Gram staining procedure and used
decolorizing solution to differentiate
structures.
- In PBS, methanol in fixation, eosin,
methylene blue, and rinse water
REGRESSIVE STAINING
- Overstain the sample then decolorize to
reduce the intensity of the color of the
stain
- Routine H&E staining because of the use
of a strong solution to stain the nucleus,
and in order to differentiate the nucleus
from cytoplasm we must decolorize it
DIFFERENTIAL STAINING
- Use of more than one chemical stain to
differentiate microorganism or structures
- Basic dyes are applied at excessive
amounts
- Decolorizer
- In H&E staining
o Nuclear(acidophilic)- blue/purple –
basophilic
o Cytoplasmic(basophilic)- red/pink
– acidophilic
- Acidophilic structure = basic structure
 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES: STAINING
- Basophilic structure= acidophilic - Ex. phagocytosis
structure - Dye used: non-toxic
(If u stain the basic structure or o Lithium
acidophilic structure with Eosin then it o Carmine
will be stained with Eosin) Eosin is an o India Ink
acidic dye - Through injection
(If u stain Basophilic structure or acid
structure with Methylene Blue or
Hematoxylin then it will take up the color SUPRAVITAL STAINING
of Hematoxylin) Hematoxylin and - Applied on extracted cells
methylene Blue is basic dye - Stains used are toxic to the cells
o Methylene blue
METACHROMATIC STAINING o Brilliant cresyl blue (Detection of
- Stains are used to stain particular Reticulocytes)
substances within the cell by giving the - Performed outside the body.
substance a color different from the color
of the stain (typically black color)
- Histological purposes, used for
demonstrating
o Cartilage
o Connective tissues
o Mucins (Alcian blue, Periodic Acid
Schiff PAS)
o Mast cell
o Amyloid

METALLIC STAINING
- Metal incorporated in stain solution are
colorless
- Reduction on the tissue produces
opacity, giving tissue a black/brown color
- Example: Silver stains and Gold stains

VITAL STAINING
- Selective staining of living cells and
cellular components
- Demonstrates cytoplasmic structures
- May also reveal certain chemicals, or
chemical reactions take place
- Intravital and Supravital

INTRAVITAL STAINING
- Injected thru bloodstream (IV-
Intravenous, IP- Intraperitonial or SC-
subcutaneous)
- Produces coloration of cells
- Through special microscopy and
processes
HEMATOXYLIN AND EOSIN STAINING
- More commonly known as the H&E
staining procedure
- STANDARD staining procedure for all
histological samples and GOLD
STANDARD for diagnosis of disease
when using tissue sample
- Provides exceptional detail that is
necessary for:
o Tissue-based diagnosis
(glycogen storage diseases; have
different classifications)
o Classification of infection,
tumors/cancer (have different
stages), metabolic disease
HEMATOXYLIN
- Derived from the heartwood of the
tree Hematoxylon campechianum
(Hematoxylum campechianum) the
common name of this tree is log wood
- Hematoxylin is not the active
component
- Not a stain itself, but the compound
HEMATEIN (active component), a -
product of oxidized hematoxylin, acts
as the dye and the one that stain basic
structures
- Anionic, but has a poor affinity to
tissue without mordants incorporated
in hematoxylin
- Mordant: important in the formulation
of different hematoxylin stains
- Mordant would include:
o Aluminium, iron, molybdenum,
lead, or tungsten salt
- Oxidation is required to produce
hematein
- Methods of oxidizing hematoxylin:
o Natural oxidation (Ripening)-
expose to light and air for 3-4
months
o Chemical oxidation- adding
certain chemicals to produce
ready-to-use stains (ex. Sodium
iodate in Mayer’s hematoxylin).
Stain may be ready for 24 hrs.
HEMATOXYLIN CLASSIFICATION
1. ALUM HEMATOXYLIN
- Most frequently used hematoxylins;
good nuclear staining particularly the
Erlich’s hematoxylin
- Mordant: Aluminum potassium
sulphate (ptash alum) or Aluminum
ammonium sulphate (ammonium alum)
- Stains nuclei red, but converts to a
blue-black color when washed with a
weak alkali solution (ex. Saturated
LiCO3, 0.05% ammonia in distilled
H2O, Scott’s tap water substitute)
o Weak alkali sol. – necessary for
us to see the blue characteristic
of nuclei of cells. Also known as
BLUING SOLUTION
- With the application of bluing solution,
the nucleus will be color blue/purple
2. IRON HEMATOXYLINS
- Uses iron as both a mordant and
oxidizing agent
- Mordant: FeCl3, and ferric ammonium
sulfate
Good for demonstrating a wider range
of tissue detail; intranuclear detail,
muscle striations, elastic fibers, myelin
- Used for tissue photography
- Over-oxidation is a problem (due to
active component of the mordant
since they also act as oxidizers)
- Should not be store for long time
3. TUNGSTEN HEMATOXYLINS
- Only one type (Mallory’s
Phosphotungstic Acid Hematoxylin
Technique)
- Mordant: 1% aqueous
Phosphotungstic acid
- Usable and stored for many years
- Applicable for both fibrin, muscle
striations and glial fibers (neurological
specimens)
- Used for tissue photography
- Mallory’s Phosphotungstic Acid
Hematoxylin Technique: helpful in
demonstrating different cellular
components that is why they are
commonly used for demonstrating
cellular detail
4. MOLYBDENUM HEMATOXYLINS - Combined with picric; produces
PICROCARMINE; ideal for
- EMordant: Molybdic acid
neuropathological studies
- Recommended for demonstrating
- Combined with aluminun chloride to
collagen, coarse reticulin argentaffin
produce BEST’S CARMINE; collagen
cells
2. SYNTHETIC DYES
5. LEAD HEMATOXYLIN
- Also called coal dyes since they were
- Mordant: lead salts
originally manufactured from
- Practical in the diagnosis and
substances taken from coal tar
demonstrating endocrine cell granules
- Derived from benzene; also
in the alimentary canal
collectively known as ANILINE DYES
6. HEMATOXYLIN WITHOUT MORDANTS - Categorized as chromogens since
simple benzene-derived stains cannot
- For demonstrating various minerals in
bind with the tissues
tissue sections (Pb, Fe, Cu)
- An auxochrome (act as glue between
- Hematoxylin can still stain different
chromophore and tissue) allows
components even there is no mordant
binding with the chromophore by
EOSIN providing the ability to form salt
- It is a fluorescent, xanthene dye bridges
- Binds with eosinophilic compounds - SUBCLASSIFIED INTO 3 GROUPS:
within cells or acidophilic compounds o ACIDIC DYES (ex. Picric acid)-
- It’s an acid dye that will stain ideal for staining collagen,
acidophilic or the basic coponents of eosinophilic granules of
cell leukocytes, and other
- Best paired up with alim hematoxylins acidophilic components
- Eosin Y- most widely used eosin o BASIC DYES - for staining
- Substitute for eosin include- phloxine, basophilic components (ex.
Biebrich scarlet Chromatin, mucus, cartilage
matrix); ex. Methylene blue
OTHER STAINS
o NEUTRAL DYES – combination
- Dyes for biological sample staining are
of acidic and basic dyes;
categorized into 2:
requires tissues to be fixed first
o NATURAL DYES- extracted from
in alcohol (ex. Romanowsky
plants or insects
stains, Geimsa, Irishman’s stain)
o SYNTHETIC DYES- or coal tar
dyes
1. NATURAL DYES
- Extracted from plants or animals
A. Hematoxylin
- exracted from tree
B. Cochineal Dyes
- Extracted from the cochineal bug
(coccus cacti)
- Treated with alum to produce
CARMINE; chromatin and nuclear
stain for fresh material
Water- ideal when sample if fixed in formalin
or aqueous solution
ROUTINE H&E STAINING SEQUENCE
- The process is applicable for tissues
fixed by most fixative, except osmic
acid
1. Deparaffinization – removal of wax; use
of (xylene 1 for 3 mins and xylene 2 for 2
mins)
2. Rehydration- use of alcohol in gradually
decreasing concentration (absolute
ethanol for 2 mins and 95% ethanol for 1-
2 mins)
3. Rinse- distilled/tap water for 1 min
4. Primary stain- use hematoxylin; stain the
nucleus red color (harris hematoxylin for 5
mins)
5. Rinse- distilled/ tap water
6. Decolorize- use of 1% acid alcohol for
10-30seconds; remove the stain of
structures that do not have an affinity to
hematoxylin
7. Rinse- distilled water is preferred
8. Blueing- making the nucleus color blue
with the use of weak alkali solution like
Scott’st’s tap water
9. Rinse- tap/distilled water
10. Counter stain (eosin)-staining structures
that are not stained by hematoxylin (1%
aq. Eosin Y for 5 mins)
11. Rinse- tap/distilled
12. Dehydrate- alcohol that is gradually
increasing (ethanol)
13. Clear- use xylene (2 changes)
14. Mount- usually aqueous (putting a
coverslip)
FROZEN SECTION STAINING
- Necessary for rapid diagnosis
- Shorter duration vs. routine H&E
staining
- Almost follow similar format f routine
H&E staining
- Other stain methods
o Thionine method –Nissl bodies
o Polychrome methylene blue
o Alcoholic pinacyanol method- SS
mitochondria; color sensitization
for photography
PAPANICULAOU STAINING
- A.k.a. pap stain; developed by George
Nicholas Papaniculaou
- Modification of H&E staining method
- Orange G 6 (OG6) and EA-50 is
incorporated
- Provides green, blue, and pink hue
subtleties to the cells
- Ideal for CERVICAL CYTOLOGY
studies
QUALITY CONTROL IN ROUTINE H&E
STAINING
- Most staining procedures are done in
bulk
- CONSISTENCY of the staining
qualities is important for preparing
histological slides
- Automation allows consistent
schedules in staining procedures
- Variability in the solution, particularly
in the hematoxylin solution, is the
most commonly encountered
- Variability is attributed to the batch
number, change of supplier, pH
difference, usage, and age
- Even if the “recipe” is followed,
variability in the staining property with
hematoxylin can and will be expected
- New batches must be checked or
efficacy against current or earlier
batches. Staining times may be
adjusted
- Fixatives, variations in the processing
schedule, thickness, and temperature
can affect staining
PRECAUTIONS WITH H&E STAINING
- Stains must not get on the skin. The
stains used are considered a health
hazard. Wear gloves if available
- Failure in staining slides may be
caused by failure to remove paraffin,
fixatives not being washed out,
decalcifying agent not washed out,
faulty stains
- Fuzzy stained sections can be caused
by old reagents (xylene, alcohol),
moisture on the coverslip (dehydrate
the coverslip), too much albumin on
the slide (prepare new slide)
- Stains may be saved and used again
as long as it hasn’t lost its staining
ability
- If a section fall to stick to the slide
after staining, this may be caused by
dirty or oily slides, fast transition in
alcohol baths, sections nor spread
well on the slide, old adhesive (use
new adhesive), signified by turbidity
and putrid odor
 CYTOLOGIC TECHNIQUES
Diagnostic cytology
- Consist of exfoliative cytology, cell
blocking of body fluids, and fine
needle aspirate biopsy
examination.
- Provide a conclusive diagnosis to a
patient
- The diagnostic value of the rest will
require that samples must include
the representative material (Ex. If
we collect synovial fluid it must
contain the essential component of
this specimen), and the method used
must give rapid results without
having to destroy most of the
cellular detail.
- Staining qualities can also affect
the interpretation of tissues
- Especial fixative methods can be
done to avoid damage
EXFOLIATIVE CYTOLOGY
- Study of cells that have been
desquamated (shaved
off/scrapped off) from epithelial
surfaces
o Normal spontaneous shedding in
the epidermis of the skin
(observed in malignancies)
o Desquamation can also be
induced by physically removing
epithelial cells (shave, swabbing,
aspiration, washings like
bronchial lining can be washed
or lavaged)
- Recommended for the following
procedures:
o Detection of malignancies;
staging cancers (neoplastic
effect can determine stage)
o Detection of precancerous
cervical lesions (pap smear)
o Assessment of female
hormonal status (sterility,
endocrine d/o)
o Determine genetic sex (detection
of Barr bodies in somatic cells)
 Barr bodies indication of
2 X chromosome
o Detection of infection
EXFOLIATIVE CYTOLOGY:
SPECIMEN PREP
1. SMEAR PREPARATION
- Must be acquired freshly (smeared
immediately); typically prepared in
the doctor’s office or radiation
units. Must be spread evenly so that
there would be minimal overlapping
of the cell
- Treated as like any other histological
slide
- Cytopreps are “wet-fixed” so that
there would be less distortion of cell
using 95% ethanol immediately after
prep
o Preserves nuclear detail; helps in
evaluating nuclear changes
o Air drying distorts the
appearance of the cell, thus it is
avoided
- Bone marrow aspirate (BMA) smears
are dried prior to fixing
o Fixatives for BMA are 95%
ethanol, pure ethanol, methanol
o Exofialtive cytology of BMA is
fixed 95% ethanol
2. CERVICAL SMEARS
- Collected in endo and ecto cervix
area (pap smear)
- Patient must do the following:
o Abstain from having sex/coitus
for at least a day since abrasions
can lead to morphological
 CYTOLOGIC TECHNIQUES
changes in the cervical lining or
vaginal canal
o Avoid douching the vaginal canal
for at least a day
o Do not apply intravaginal
preparations for 1 week prior to
the procedure
- For collecting specimens:
o Collection should not be done
during menstrual bleeding
because RBC can interfere to
the value
o Collection systems are available
for collecting sample
- Collection systems:
o Cotton swabs – common but
highly discouraged if alternatives
are available because of the
fibers in the cotton swab may
interfere with the preparation of
the smear
o Wooden spatula – highly
recommended because of its
mildly rough surface allows for
more material collection
o Cytobrushes – induced
abrasion to the cervical canal;
gynecological cell specimens;
blood may be present
o Endocervical brush – has
especial shape; strictly for
collecting endocervical and
ectocervical material
Cytoprep:
If you want to preserve sample
place/suspend it in a fixative that is
present in ThinPrep and SUREpath
method
3. IMPRESSION SMEAR
- Often done on ulcerated surface
lesions
- Allows immediate assessment of
lesion before fixation and processing
- Also indicated for assessing tumors,
particulary the lymph nodes
4. SPUTUM SMEARS
- Collected at least 3 consecutive
morning sputum specimens since
the have higher conc. of bacteria
- For bacterial infection
- Sputum specimen is collected in a
wide-mouthed container containing
Scaccomanno’s fluid (50% ethanol
+ 2% carbowax)
- Induced sputum expectoration can
be performed when patient cannot
expel sputum
o Inhalation of aerosol solution for
20 minutes
- A minimum of 2-4 slides are
necessary for extensive study of
sputum
- Can be stain with H&E, pap smear,
acid fast slide for acid fast bacilli like
M. tuberculosis, gram staining for
diagnosis of bacterial infection of
lower respiratory tract
- Solid particles are set aside (ex.
Blood). Crush preps are created out
of these
- Thick and thin smears out of the
sputum sample is created. Wooden
applicator sticks aid in spreading
the sample evenly
Specimen is fixed immediately in
95% ethanol (wet prep) immediately.
5. BRONCHOSCOPY SPECIMEN
 CYTOLOGIC TECHNIQUES
- Bronchial brushing are directly
smeared on slides via pull technique
- Slides are fixed in 95% ethanol or
with a spray fixative
- Washing are collected in containers
and cytospin preps (suspension
made of diff swabs then centrifuged)
are made these can be created in a
cell block and the same process on
conventional tissue processing
- Aspirates are collected either via
glass suction apparatus or by
washing or lavages (1-2 mL saline)
- Indication of a good collection:
presence of CILIATED BRONCHIAL
CELLS
6. GASTRIC SECRETIONS AND
ASPIRATES
- Collected by simple irrigation and
aspiration technique
- Examined ASAP; delays or more
than ½ hour before fixing can cause
cell digestion
- Patient should have fasted for at
least 8 hours
7. BREAST SECRETIONS
- Nipple discharges provide
extremely low diagnostic value for
diagnosing breast carcinoma
- Nipple discharges are caused by:
o Hormonal imbalance is the main
cause of spontaneous
discharging in young patients
o Blood in the discharge indicates
benign intraductal papilloma
- Discharge should be smeared on a
clean glass slide and immediately
fixed (95% isopropanol or spray
fixative)
FINE NEEDLE ASPIRATION
- Study of cellular samples of organs
that do not shed cells:
o Breast
o Thyroid
o Lymph nodes
o Liver
o Lungs
o Skin (subcutaneous)
o Soft tissues
o Bone
- For assessing easily palpable lesions
- A 25 gauge needle equipped to a 10
mL syringe is used. Lesion is
repeatedly probed while a small
amount of suction is applied
- Slides are prepared and is air-dried
as quickly as possible to reduce
shrinkage
- Tools such as laparoscopy,
computerized tomography (CT
scan) or ultrasound (sonography)
can aid in collecting deeply seated
lesion
- Sonography common in FNAD
- The first few drops provide the most
diagnostic material.
BODY FLUID MICROSCOPY
- Cytospin preparation are
considered the best for concentrating
cells in different body fluids
o Cerebrospinal fluid (CSF)
o Serous fluids (peritoneal,
pericardial, pleural)
o Urine
- Body fluid analysis can cast light to
the origin and type of primary
tumors
CYTOLOGIC TECHNIQUES
 CYTOLOGIC TECHNIQUE’S PART 2
PAPANICOLAOU SMEAR STAINING o Crisp blue to black-stained
nucleus
 (Develop by Dr. George Nicholas
o Highly keratinized cells and cells
Papanicolaou) with high glycogen content are
 A.K.A. Pap smears is still considered to yellow
be method of choice for exfoliative o Superficial cells are orange to
cytology; the “gold standard” pink
 Routine staining procedure for o Intermediate and parabasal cells
cytopathological studies. (Cervical or are turquoise green to blue
vaginal) o Metaplastic cells are often
 Polychrome staining technique that stained both green and pink at
results in a well-stained nuclear once.
chromatin, differential cytoplasmic
staining, and cytoplasmic transparency.
ADVANTAGES:

PRINCIPLE: 1. High alcohol content of cytoplasmic

counterstains allows the overlapping of
 The NUCLEAR STAIN in the cells to still to be seen and identified.
procedure is HEMATOXYLIN (Harris 2. Nuclear detail is excellent
Hematoxylin) (nucleus-purple color 3. Color range is predictable and has a
after bluing effect), which acts as the high diagnostic value in identification
PRIMARY STAIN, while the and classification of cells.
CYTOPLASMIC STAINS are OG-6 4. Valuable in comparing cellular
and EA-36/50. (They would replace appearances.
Eosin in the routine H&E staining
technique in order to modify, produce
pap smear staining technique) USES:
 The first counter stain (OG-6) stains
keratin. 1. Gynecological smears (pap smears)
 Second counter stain would be (MAIN SPECIMEN)
Eosin Azure dye its either EA 36/50. 2. Sputum
 The Eosin Azure stain is composed 3. Brushings
of 3 stains: 4. Washings
o Eosin Y- superficial epithelial 5. Urine
squamous cells, nucleoli, cilia, 6. CSF
RBC’s 7. Serous fluids,
o Light Green SF- cytoplasm of 8. Synovial fluid
other cells (Fast Green FCF 9. Seminal fluid
can be used as substitute) 10. FNA material
o Bismarck Brown Y stains 11. Tumor touch samples
nothing. (Sometimes omitted) 12. Other cytological preparations
 Stained specimen display hues of the
entire spectrum (red to violet)
 A well-prepared specimen has the
following characteristics:
 CYTOLOGIC TECHNIQUE’S PART 2
 Found during the 1st-10 day post
menstruation.
CELLS IN A CERVICAL AND VAGINAL
 Shed in response to ovarian hormones
SMEAR
 Marker for unsatisfactory or an
MATURE SUPERFICIAL CELLS unreliable smear (if mixed with WBCs
and RBCs).
 Polygonal squamous cells
 Pale, pink-staining cytoplasm ENDOCERVICAL GLANDURAL CELLS
 Dark pyknotic nucleus
 Occur in large groups or small sheets
INTERMEDIATE CELLS  Pale blue/gray cytoplasm; finely
vacuolated
 Polyhedral or elongated
 Indistinct cell borders
 Basophilic vacuolated cytoplasm
 Nuclei contains finely granular chromatin
PARABASAL CELLS  Presents a HONEYCOMB
APPEARANCE when viewed on end.
 Round or oval cells
 Small, dense, basophilic cytoplasm
 Large vesicular nucleus CELLS IN A CERVICAL AND VAGINAL
 Normally found two weeks of age to SMEAR
puberty, after childbirth, with
abortions and after menopause  These different cells would give an
image as to what happens inside the
NAVICULAR CELLS cervix and vaginal canal, providing an
 Boat-shaped intermediate cells insight into certain conditions:
 Folds or curls on edges o Cell maturity- influenced by
 Presence suggests combined estrogen release
estrogen-progesterone effect o Hallmark of atrophy in
 Seen in the latter half of the menstrual postmenopausal women-
cycle, during pregnancy, and indicated by basal and
menopause parabasal cells
o Squamous metaplasia (common
PREGNANCY CELLS in the endocervix)- parabasal
cells
 Round, oval, or boat-shaped cells
o Hyperkeratosis – anucleate
 Translucent basophilic cytoplasm
mature polygonal squamous
(glycogen accumulation)
cells
 Nucleus is pushed to the side or o Reactive conditions (cervicitis) –
towards the membrane presence of conspicuous nucleoli
 Deeper blue stain at the cytoplasmic in endocervical cells
periphery o First 12 days of the menstrual
ENDOMETRIAL CELLS cycle – endometrial cells

 Small cells; slightly cylindrical

 Less basophilic cytoplasm
 Tightly packed groups of 3 or more
cells
 CYTOLOGIC TECHNIQUE’S PART 2

INFECTIONS IN THE CERVICAL AND Actinomyces spp.

VAGINAL CANAL
 Associated with females implanted with
 Infection on the site can also be IUD.
detected by:  Tangled clumps of bacteria,
o Detecting the offending resembling dark cotton balls or dust
microorganisms (bacteria, bunnies.
fungi, parasite)
 Stained via the Pap
staining procedure.
o Cytopathic effect (CPE) (seen in
viral infections)

INFECTIONS ON CERVICAL AND VAGINAL

CELLS
Gardnerella vaginalis and other bacterial
infections
 Presence of clue cells
 Multiple short bacilli (coccobacilli),
curved bacilli, or mixed bacteria
morphology
 Absence of Lactobacillus spp. Bacteria
 Filmy smear appearance

Trichomonas vaginalis
 Present in sexually active patients
presenting a burning, itching sensation.
 Found outside the epithelial cells
 May be confused for a WBC

Candida spp. Infections

 Candida spp. (C. albicans, C. glabrata)
infects the vulva, vagina, and the cervix.
 Patients often present thick, cheesy
discharges
 Eosinophilic staining property
 Often found in its yeast forms, forming
“shishkebab” formation of squamous
cells