TISSUE PROCESSING

The purpose of processing a tissue is to provide a solid support medium for tissue during section cutting.
This can be achieved by passing tissue through a series of reagents, which end in a solid medium. If tissue is
not supported with a solid medium, thin sections cannot be cut. Light is also necessary for microscopy and
only thin sections permit light to pass through them. Tissue processing involves various stages between
fixation and cutting of sections. The stages are:
 Dehydration
 Clearing
 Infiltration
 Embedding
The procedure in these stages do not apply to frozen section
SELECTION OF TISSUE FOR PROCESSING
Following fixation, pieces of tissue for histological examination are selected from the gross specimen. A
brief description of the nature of the tissue and site of origin should be recorded. The tissue block taken can
be up to 3×2.5cm in area and about 4mm thick, are placed in a perforated cassette. The introduction of
plastic embedding cassettes has greatly facilitated the processing of tissue and reduced the risk of possible
error. These cassettes have sides where necessary information can be recorded in pencil. The cassettes form
part of the final paraffin wax blocks, which means that the tissue is always identified.
DEHYDRATION.
Dehydration is a process of removing water from tissue. Graded solutions of alcohol are used. The
concentrations range from 70% to 100% (absolute) alcohol. Tissue has to be passed through low
concentrations of alcohol to high concentration because when water molecules mix with absolute alcohol,
there is usually turbulence at point of contact, which can distort tissue constituents. Tissue must be allowed
sufficient time in absolute alcohol to enable complete dehydration. If dehydration is not complete, the
clearing agent and wax will not penetrate tissue. This will lead to poor section cutting. The solutions
commonly used to carry out dehydration are: ethanol (ethyl alcohol), methyl alcohol, iso-propyl alcohol,
industrial alcohol (methylated spirit), rebutanol, tertiary butanol, polyvinyl alcohol, acetone and Dioxane.
Dehydration is important because most embedding media are not miscible with water, and the removal of
water facilitates the subsequent impregnation with the embedding media.
The Alcohol Method
The best agent for dehydration is ethyl alcohol which has the advantage of not being poisonous. Dehydration
should be more gradual, beginning with 70% alcohol, with smaller intervals between the different grades of
alcohol. To check for the presence of water in the final bath, add a small amount of dried white copper
sulphate. If water is present, the copper salt will show a tinge of blue. Alternatively, a layer of anhydrous
copper sulphate covered with filter paper is kept at the bottom of the jar used. The salt acts as an indicator,
turning blue when water is present. When this happens, the alcohol should be replaced. It is essential to use
high quality top grade alcohol e.g Ethanol. In the absence of a high grade alcohol, isopropanol is the next
choice of alcohol for dehydration. It has been claimed by some researchers that the time necessary for
dehydration may be shortened when processing is carried out at 37°C instead of at room temperature. This
makes the method ideal for urgent work.
The Acetone Method
Acetone may be used and has been found to be a good dehydrant, cheap but very volatile. It hardens tissue
more than alcohol and greater volume of it is required than alcohol. Dehydration takes from 30 minutes to 2
hours with resultant shrinkage, and for this reason it is not quite suitable for routine work. Only small pieces
of tissues should be treated in acetone.
The Dioxane Method.
Dioxane is miscible with water, alcohol, xylene, balsam and paraffin wax. This makes it a unique
dehydrating agent. There is very little shrinkage to the tissue. But it is not recommended for routine use
because it is a dangerous substance with little warning odour and a cumulative toxic action.
 CLEARING
This stage of tissue processing can conveniently be referred to as de-alcolhalisation- the removal of alcohol
from blocks or sections of tissue by immersing them in an ante-medium. In addition to removing the alcohol
from the tissue, most of well-known ante-media have the property of making the tissues transparent. This
stage of tissue processing is referred to as clearing stage and the anti-media as clearing agents. It should be
pointed that some ante-media (e.g chloroform) have no clearing property. Clearing fluids should be used in
amount not less than 10 times the volume of the tissue. Common clearing agents are Xylene, Toluene,
Benzene, chloroform, Histo-clear and cedar wood oil.
Xylene
This is a rapid clearing agent, suitable for urgent biopsies. It is highly flammable, tissues are rendered
transparent by xylene and it volatilises readily in the paraffin oven. Tissues should never be left in xylene for
more than 2 hours. Biopsies and tissue blocks not exceeding 3mm in thickness are cleared in 15 to 30
minutes, some materials, notably brain and blood-containing tissues, tends to become brittle if immersion in
xylene is prolonged. When dehydration is not complete, xylene becomes milky when the section or tissues
are immersed into it. Xylene is cheap and is usable with celloiding sections
Toluene
It is highly flammable; it is somewhat more expensive than xylene. Toluene is slower than xylene in action,
clearing time is from 15minutes to 2 hours, depending on the tissue type and thickness. In general properties,
toluene is similar to xylene.
Benzene
It has properties very similar to xylene. It is not recommended for usage as it is known to be carcinogenic.
Chloroform
An expensive, non-flammable clearing agent. It is the most ideal clearing agent for the central nervous
system tissues, lymph nodes and embryos because it causes little shrinkage and does not harden tissues
excessively. It is good for large blocks; it is not a classical agent as it does not render tissues transparent. De-
alcoholisation time for tissues 3mm to 5mm thick is 5 -20 hours. Precautionary measures are necessary when
handling chloroform as its vapour is toxic and anaesthetic. It must be used in a well-ventilated room and in a
tightly closed container.
Cedar wood Oil
It is rarely used for routine clearing purposes because of its cost and slow action. It is ideal for research work
especially in the study of embryos. This reagent imparts proper consistency to dense fibrous tissues and skin
tissues which allows easy cutting of the sections. It also imparts transparency to the tissues. One major
disadvantage is the difficulty in eliminating the oil from the wax oven
Other Clearing Agents
Other agents that can be used but are not of great importance are carbon tetrachloride, carbon disulphide,
paraffin oil and clove oil.
Infiltration
The stage can also be called Impregnation. It is a process of replacing a clearing agent with molten paraffin
wax. The paraffin wax completely displaces the clearing agent from the tissue and it is replaced by the
paraffin wax. Infiltration is faster if it is done at a reduced pressure in a vacuum oven
Vacuum Impregnation
This involves rapid replacement of clearing agent with paraffin wax at a reduced pressure. It also aids
removal of trapped air bubbles from tissue. Vacuum impregnation is particularly useful for urgent biopsies
and lung tissue because of the pocket of air which lung contains. In this technique, dehydrated and cleared
tissues are put in paraffin wax in a vacuum oven set at a temperature of 2°C to 3°C above the melting point
of the wax used. The vacuum oven is connected to a water pump or an electric pump which evacuates air
gradually from the oven until the pressure in the oven is reduced to about 300mmHg to 500mmHg. Tissue is
passed through two changes of paraffin wax in the oven but at half the normal time. Tissue is then removed
from oven and embedded in paraffin wax.
 Embedding
Embedding is a process of burying a tissue in molten paraffin wax. The paraffin wax becomes a solid firm
structure when it is cold. This forms a support medium for the tissue during microtomy. Tissue should be
well oriented in the moulds during embedding so that a complete representation of all parts of the tissue is
present in cut sections. Embedding is performed in special containers called moulds. These containers help
to give shape to the wax containing the tissue when it is set.
Embedding Moulds
Embedding moulds can also be called blocking out moulds. They are rigid materials into which tissue and
molten paraffin wax are put so that when wax solidifies, it forms a solid mass, which takes the shape of the
mould.many types of embedding mould are available, these include: Leuckhart embedding box, Plastic ice-
cube tray, tissue tek metal base mould.

An 18 hour Automatic Tissue Processing

Beaker 1 10% formol saline 1hour 30 min
Beaker 2 70% alcohol 1 hour
Beaker 3 80% alcohol 1 hour
Beaker 4 90% alcohol 1 hour
Beaker 5 95% alcohol 1 hour
Beaker 6 95% alcohol 1 hour 30 min
Beaker 7 Absolute alcohol I 2 hours
Beaker 8 Absolute alcohol II 2 hours
Beaker 9 Xylene I 1 hour 30 min
Beaker 10 Xylene II 1 hour 30 min
Wax Bath I 2 hours
Wax Bath II 2 hours

The processing time given above varies with type of tissue and type of tissue processor. The time can be
reduced or increased depending on when the Laboratorian is ready for embedding of tissues.

Routine Manual Tissue Processing Method

Note: tissue should not be more than 4mm thick
1. Wash fixed tissue in water if necessary
2. 70% alcohol 1-6 hours
3. 90% alcohol 1-6 hours
4. 95% alcohol 1-16 hours
5. Absolute alcohol- 2 changes 2 hours 30 min each
6. Chloroform or Cedar wood oil About 16 hours
7. Xylene or Toluene 1 Hour
8. Paraffin wax -3 changes 1 hour each.
Embedded in paraffin wax
Where chloroform or cedar wood oil is not available, tissue may be left in the second bath of absolute
alcohol overnight and treated with two changes of xylene for 1 hour 30 min each.
Then continue from stage 8.
Tissue should not be left in xylene overnight because prolong exposure of some tissues to xylene makes
them brittle.