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 agentsCon
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

soluble in processing fluids

suitable for sectioning and ribboning
molten between 30C and 60C
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 39C to

Paraffin waxCon
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

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

Paraffin InfiltrationCon
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 (58-60C) 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 heatinduced tissue hardening, facilitates
complete removal of transition
solvents, and prolongs the life of wax
by reducing solvent contamination.

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

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(56-58C) ------ 1
hr
b-Paraffin-II ------------------- 1
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.

EmbeddingCon
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.

EmbeddingCon
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

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.

EmbeddingCon
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