Dehydration
Dehydration
• Tissue fixed in aqueous fixatives have high
water content. The process of removal of this
water so –called (free water), i.e., water not
bound to thee tissue components but within
the spaces of the protein network, is termed
dehydration.
Dehydration
- Routine dehydration is accomplished by
using a serious of gradually increasing
percentage
of ethyl alcohol to reduce
shrinkage and distortion of tissue
components.
Dehydration
• Alcoholic series and time in each percentage
depend on tissue size and type of tissue.
• The volume of alcohol should be at least 10 times
the size of the tissue.
• The percentage of alcohol usually employed are
70, 70, 95 and 100%, where the tissue is placed
into two or three changes for 1 hr each.
• For special, delicate or large pieces of tissue,
dehydration should start at either 30% or 50%
and might remain for longer than 1hr in each
concentration.
Types of dehydrating agents
a- Ethyl alcohol (nontoxic)
b- Dioxane
C- Cellosolve
d-others (methyl alcohol, acetone, isopropyl
alcohol)
Clearing
Clearing (dealcoholization)
Introduction
• This is the step between dehydration and
paraffin infiltration.
• The purpose of this step is removal of the
alcohol and replacement with a fluid that will
mix with paraffin.
• The tissue is placed in two or three changes of
clearing agents (volume 10 times the tissue).
Clearing (dealcoholization)
Introduction
• Good clearing agents should replace alcohol
quickly and not over harden the tissue.
• This step is called clearing because they
penetrate, make the tissue transparent and
also harden it.
• Too long in the clearing agent hardens the
tissue and the tissue become difficult to
section.
Clearing agents
• Most clearing agents are highly toxic and
inflammable and should be handled with care.
• Xylene is the most commonly used clearing
agent. If dehydration is incomplete the xylene
becomes opaque or milky as water comes out of
the tissue. If this occurs, put the tissue back into
absolute alcohol and continue dehydration.
• Benzene penetrates and clears tissue very rapidly,
produces a minimum of shrinkage.
• chloroform are also clearing reagents.
• Cedarwood oil.
Clearing agents…Con
• Choice of clearing agents depend upon the
following:
1- The type of tissue to be processed.
2- Speedy removal of the dehydrating agent.
3- Ease of removal by molten paraffin wax.
3- Minimal tissue damage.
Infiltration
Infiltration
• Is the saturation of tissue cavities and cells by
a supporting substance which is generally, but
not always, the medium in which they are
finally embedded.
• Is the process of replacing clearing agent by
embedding media.
• Tissues are infiltrated by immersion in a
substance such as a wax, which is fluid when
hot and solid when cold.
Infiltration
• Alternatively, tissues can be infiltrated with a
solution of a substance dissolved in a
solvent, for example nitrocellulose in
alcohol-ether,
which
solidifies
on
evaporation of the solvent to provide a firm
mass suitable for sectioning.
Ideally an infiltrating and
embedding medium should be
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soluble in processing fluids
suitable for sectioning and ribboning
molten between 30°C and 60°C
translucent or transparent; colourless
stable
homogeneous
capable of flattening after ribboning
non-toxic
odourless
easy to handle
inexpensive
Paraffin wax
• PROPERTIES
Paraffin wax is a polycrystalline mixture of solid
hydrocarbons produced during the refining of coal
and mineral oils.
• Wax hardness (viscosity) depends upon the
molecular weight of the components and the
ambient temperature. High molecular weight
mixtures melt at higher temperatures than waxes
comprised of lower molecular weight fractions.
Paraffin wax is traditionally marketed by its melting
points which range from 39°C to 68°C.
Paraffin wax…Con
• Tissue-wax adhesion depends upon crystal
morphology of the embedding medium.
Small, uniform sized crystals provide better
physical support for specimens through close
packing. Crystalline morphology of paraffin
wax can be altered by incorporating additives
which result in a less brittle, more
homogeneous wax with good cutting
characteristics.
Paraffin Infiltration
Properties of paraffin
Is rapidly converted from solid to liquid form
on heating.
permeates the tissue in a liquid state.
solidifies relatively quickly on cooling.
When the paraffin solidifies it becomes firm
enough to section at room temperature.
MODIFIED PARAFFIN WAXES
• The properties of paraffin wax are improved for
histological purposes by the inclusion of substances
added alone or in combination to the wax:
- Improve ribboning: prolong heating of
paraffin wax at high temperatures or use
micro-crystalline wax.
- Increase hardness: add stearic acid.
- Decrease melting point.
- Improve adhesion between specimen and wax.
Paraffin Infiltration…Con
• Once the liquid paraffin has infiltrated
hardened, it maintains the components of the
tissue in proper relation to each other;
otherwise these components would be
compressed and distorted during sectioning.
Advantage of paraffin infiltration
1- Time of infiltration and embedding are
short.
2-Thin sections can be cut with the rotary
microtome and sections will adhere to each
to form a ribbon.
3-Tissue once infiltrated and embedded can be
stored in a dry condition indefinitely
without damage to the tissue.
Paraffin Infiltration
• Infiltration proceeds by placing the tissue
in at least two or three changes of liquid
paraffin in the paraffin oven (58-60°C) for
a total of 2-4hr.
Vacum infiltration
• is the impregnation of tissues by a molten
medium under reduced pressure. The
procedure assists the complete and rapid
impregnation of tissues with wax, reduces the
time tissues are subjected to high
temperatures thus minimising heat-induced
tissue hardening, facilitates complete removal
of transition solvents, and prolongs the life of
wax by reducing solvent contamination.
Vacuum infiltration requires a vacuum
infiltrator or embedding oven
manual technique
- The usual method for processing tissue
is to pass the tissue from one fluid to
another. It needs the person physical
appearance.
- To avoid handling of the tissue with
forceps use the method of decantation.
- In this method the tissue is in one
beaker only and the fluids are added to
and poured off from the beaker.
Routine timing schedule for manual
technique
1-fixation : 12-24 hr
2-Wash : uses running tap water (6-8 hr)over night.
3-Dehydration: 70% alcohol-------------1hr
80%alcohol--------------1hr
95%alcohol--------------1hr
100%alcohol I-----------1hr
100%alcohol II----------1hr
4-Clearing
a-clearing agent I--------------1hr
b-clearing agent II--------------1hr
Routine timing schedule for
manual technique
5-Infilteration(in paraffin Oven)
a-Paraffin-I(56°-58°C) ------ 1 ½ hr
b-Paraffin-II ------------------- 1 ½ hr.
6-Embed
Automatic tissue processing
• Tissue can be rapidly processed without any
attention on the part of the technician from
fixative through infiltration by automatic tissue
processors (histokinetic machine). In the
machine the tissue is forwarded automatically to
the next step after specified period of time.
• The most recent automatic tissue processor use
vacuum, vertical oscillation and heat to rapidly
promote chemical activity, penetration and
interchange of the various solutions used in
processing of the tissue. Every step is adjusted for
specific period of time by using a time device.
Embbeding
• After the infiltration, biological tissue must be
supported in a solid medium or hard matrix,
firm enough to support tissue and give it the
sufficient rigidity to allow thin sections to be
cut, and yet soft enough not to damage the
knife or tissue.
Embedding materials
• Various embedding materials such as:
- Paraffin wax.
- Agar.
- Gelatin.
- Celloidin.
Embedding…Con
• During this process tissue samples are placed
into molds along with liquid embedding
material which is then harden. This is achieved
by cooling (with most embedding materials).
• The hardened blocks containing the tissue
samples are then ready to be sectioned.
Embedding…Con
• Paraffin wax is most commonly used as
embedding material. The properties of
paraffin wax are improved for histological
purpose by adding other substances to:
- improve ribboning.
- increase hardening.
- decrease melting point.
- improve adhesion between specimen and
wax.
Precautions while embedding in
paraffin wax
• The wax is clear from clearing agent.
• No dust particles must be present.
• Immediately after tissue embedding, the wax
must be rapidly cooled to reduce the wax
crystal sizes.
Mold systems
• There are four mold systems present for use:
1- Traditional method using paper boats.
2- Metal or iron container.
Mold systems
3- The peel – a- way system using disposable
plastic molds.
4- System using embedding rings or cassettebases which become an integral part of the
block and serve as holder in the microtome.
Orientation of the tissue in the
block
• Correct orientation of the tissue in the
mold is the most important step in
embedding.
• Incorrect placement of tissues may result
in diagnostically important elements
being missed or damage during
mirotomy.
Orientation of the tissue in the
block
• Elongate tissues are placed diagonally across
the block.
• Tubular and walled tissues s as to provide
transverse sections showing all tissue layers.
Orientation of the tissue in the
block
• Tissue with an epithelial surface such as skin,
are embedded to provide sections in a plane
at right angles to the surface.
• Multiple tissue pieces are aligned across axis
of the mold and not placed at random.
Embedding…Con
• During cooling, paraffin wax shrinks up to
15%, causing compression in tissues. This
compression is almost fully recovered when
sections are floated on a warm waterbath6;
compression resulting from microtomy is only
partially recovered