TISSUE PROCESSING

The examination of tissues with a microscope requires very thin slice of tissue that can transmit
light. The preparation of the thin section is called microtomy. Before such tissues are sliced or
sectioned, they must undergo a preparatory treatment through various reagents entailing
infiltration of the tissues with a suitable embedding medium which provides internal and external
support to the tissues.

The embedding medium must be of good consistency for easy microtomy. This preparatory
treatment of tissues before sectioning is called tissue processing. The processing method
employed is named after the embedding medium used. There are many embedding media used in
histology, each with it’s special advantages and limitations. The chief examples include the
following :-

1. Paraffin wax embedding media.

2. Ester wax embedding media.
3. Celloidin.
4. Gelatin.
5. Synthetic resins
6. Polyethylene glycose

The recommended tissue processing method is that which uses the paraffin wax. It is used for
routine work and even research work. Tissue processing with this method involves the following
stages in the order given :-

1) Dehydration
2) Clearing
3) Infiltration (internal support).
4) Embedding/Blocking (external support).

1. DEHYDRATION

This is the removal of water from the tissues. The water may have been introduced into the
tissues during fixation in addition to natural water. Water may also have been introduced during
washing of the tissue. These waters are removed by use of a suitable dehydrants. The common
dehydrants used during dehydration include alcohols such as;

-ethyl alcohol
-isopropyl alcohol
-methyl alcohol
-butyl alcohol e.t.c.

The other dehydrants are :-

a) Acetone
b) Dioxane (Diethylene Dioxide)
c) Pyridine
d) Cellosolve.

The reason of removing water by a dehydrant from a tissue is because the water is immiscible
with molten paraffin wax which will not penetrate the tissue in the presence of water. Dehydration
is accomplished by passing the tissues through a series of graded alcohols to avoid rapture or

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sudden shrinkage which would occur if the tissues were placed in an absolute alcohol. The
recommended concentration of the first bulk of alcohol for very fragile tissues is 30% alcohol.
70% alcohol is recommended for most adult tissues, (fragile tissues include those of embryos,
cysts e.t.c). It is important tissues be given enough time in each alcohol whatever the
concentration. Insufficient dehydration will automatically lead to improper infiltration.
Consequently, section cutting will present difficulty with that tissue .In practice, dehydration may
be carried out by hand manually or by automatic tissue processing machines. The most common
alcohol range of concentration used is 70%, 90% and 3 – 4 changes of absolute alcohol.

NB – the entry point of tissues fixed in Bouin’s, Carnoy’s, Susa fixatives will be above 70%
alcohol concentration i.e. 90% or 95%. To ensure that the bath with absolute alcohol remains
clear of water, a layer of anhydrous copper sulphate about ¼ inch is put on the bottom of the
container to act as an indicator turning blue when water contaminants are introduced. When the
anhydrous copper sulphate turns blue, change alcohol or the most economical manner is discard
the and shift all other baths backwards one step and refill the absolute alcohol afresh. Acetone -
can be used as a dehydrant only for urgent tests. It causes much shrinkage as it acts fast.

Dioxane

Although miscible with both water and wax it is unsafe in the lab for it’s toxic vapour. It’s also
very expensive to buy and also tissues tend to fall off from the wax block.
i.e limitations of Dioxane
a) unsafe due to it’s toxic vapour
b) very expensive to buy.
c) Tissues tend to fall off from the wax block.

DEHYDRATION PROCESSING SCHEDULE

Ideal tissue piece size is 3 – 5 mm.

ROUTINE AUTOMATIC PROCESSOR (12 BEAKERS)

1 10% formal saline 2hrs ] fixing

2 70% alcohol 2hrs

3 90% alcohol 2hrs
4 95% alcohol 1hr Dehydration
5 100% alcohol (Analar) 1hr
6 Analar (100%) II 2hrs
7 Analar (100%) III 2hrs

8 Equal parts of 100% 1hr

chloroform
9 pure chloroform I 1hr clearing
10 Pure chloroform II 2hrs

11 Molten paraffin wax 3hrs

12 Molten paraffin wax 3hrs Infiltration

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MANUAL PROCESSING

1. Fix up to 24hrs
2. Wash if necessary
3. 70% alcohol 3 – 8hrs
4. 90% alcohol overnight
5. Absolute alcohol 2hrs
6. Absolute alcohol 3hrs
7. Absolute alcohol III 3hrs
8. Chloroform overnight
9. Molten paraffin wax (3 changes) 3 hrs each

10. Block in fresh paraffin wax.

NB i) Vacuum infiltration is recommended.
ii) Tissues must be blocked after infiltration
iii) Tissues which require attachment to a wooden holder may need trimming of the block to
suit
the size of the wooden block being used.
iv) A sharp scapel knife must be used. The edges of the wax block must be parallel and a wax
margin of about 3-4 mm must be left on all the edges of the tissue.

2. CLEARING

This stage is also called de-alcoholization. This is the stage whereby alcohol is removed from the
tissues by a reagent that is miscible with it.
Definition – clearing is the process of removing alcohol from the tissue by a reagent that is
miscible with it during tissue processing.
The treatment of tissue with a clearing agent makes the tissue more or less transparent
(translucent) because of the increased “ refractive index” of the tissue. Most clearing reagents
used in paraffin wax tissue processing methods are miscible with alcohol and also with wax
which displaces it. Many clearing reagents are available each with it’s applications and
limitations. Most of them are volatile, toxic, flammable. An exception is chloroform which is not
flammable but is volatile and toxic hence great care should be taken when handling these
materials in regard to safety. The clearing reagents commonly used include the following :-

a) Xylene
b) Toluene
c) Benzene
d) Chloroform
e) Ceder wood oil
f) Carbon disulphide
g) Clove oil
h) Methyl benzoate

3. INFILTRATION

It is the treatment of tissues that have been dehydrated and cleared by using molten paraffin wax.
During this stage the molten paraffin wax penetrates into the tissue to provide internal support
when the wax solidifies. The molten paraffin wax also displaces the clearing reagent used during
clearing. To achieve complete removal of the clearing agent, three to four changes of the molten
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paraffin wax may be required. The temperature of the molten paraffin wax used in infiltration
must be maintained at 2 - 3 degrees Celsius above the melting point of the wax. Paraffin wax is
usually the embedding medium of choice for infiltration. It is produced during petroleum oil
processing.

The best histological paraffin wax is filtered during the course of it’s preparation but, it may be
re-filtered before use in the laboratory using Whatman’s filter paper number one (NO.1). the
hardness of paraffin waxes with melting points of 45 – 54 degrees Celsius are regarded as soft
waxes , and those with melting points of 55 – 60 degrees Celsius are regarded as harder waxes.

A) OVEN INFILTRATION

Easter wax is recommended for this. It has a melting point of 45 – 48 degrees Celsius and it sets
to become harder than the recommended routine paraffin wax of melting point 56 degrees
Celsius. Some substances can be added to the ordinary paraffin wax to improve it’s consistency
and cutting quality of the block. Examples of improved waxes are of improved paraffin wax:-

a. Parablast
b. Firmway

Substances which have been added include synthetic rubber and plastics. The addition of these
synthetic rubber does not change the melting point of the wax. These improved waxes have the
following advantages over the ordinary paraffin wax :-

1. They have superior cutting quality.

2. Good ribonning of sections.
3. No crystal formation on solidification.
4. No wringing of sections during cutting.
5. Wax sets become harder than the ordinary wax.

The molten paraffin wax for infiltration and blocking /embedding must be kept in electrically
heated, thermostatically controlled oven or wax baths or in water jacketed incubators. The
presence of water vapour in the oven or bath should be avoided because it will inhibit proper
infiltration of the wax into the tissues.

The infiltration process is carried out by immersing properly cleared tissues in the molten
wax. The number of changes of the fresh molten wax and the duration required for each bath will
depend on three factors, these are :-

1. The density of the tissue being infiltrated.

2. The size of the tissue being infiltrated.
3. The clearing agent that was previously used

NB i) Manual infiltration takes up to 9hrs whilst vacuum infiltration can take up to 3hrs (shorter time).
ii) Higher temperatures in infiltration (chambers) must be avoided (higher than 60 degrees Celsius),
because this his will cause shrinkage and hardening of tissues. Even higher temperatures will cause
cooking effect and crumbling of tissues.
iii) Clearing reagents such as cedar wood oil and chloroform are difficult to displace completely
with molten paraffin wax. It is however advantageous to give the tissues a 30mins bath in a
mixture of equal volumes of molten paraffin wax and clearing agent before transferring the tissue
into a pure bath of molten paraffin wax

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Other clearing agent –are the cedar wood oil or chloroform.

iv) Over-treatment of tissues with molten paraffin wax must be avoided. Inadequate treatment
with molten paraffin wax must also be avoided. Inadequate infiltration of the tissue will result in
the shrinkage of the tissue and the tendency of the tissue to fall off from the paraffin wax block or
crumbling during sectioning.
v) Infiltration of tissues can also be done in a vacuum oven or vacuum chamber. This has the
advantage over paraffin wax oven infiltration in that the time taken to complete infiltration is
drastically reduced usually to half an hour (30min).

B) VACUUM INFILTRATION TECHNIQUE.

Depends upon the production of negative pressure inside the embedding oven. The pressure
reduction hastens the extrusion of air bubbles and of the clearing agent from the tissue block
facilitating rapid penetration by the wax.

Tissue is impregnated(inside spaces filled and outside coated) with paraffin wax to provide
internal support .
It is useful for the following tissues :-
a. Urgent biopsies
b. Dense tissue
c. Lung tissue
d. Tissue which contain a large amount of fat.

VACUUM INFILTRATION OVEN

There are several types of vacuum impregnation (or vacuum impregnation oven)

Vacuum infiltration oven

See the diagram below

Method of use:
a. Transfer the cleared tissue to a container of molten paraffin wax and place it in a vacuum .
b. Place the heavy glass lid in position and press it firmly down.
c. Close the valve and exhaust the chamber with the venting pump.
Shown on the manometer.
d. Close the stop – clock (clip) between the Y piece and the trap bottle and then turn off the
pump.
e. When the tissue has been immersed in the wax for the requisite period, unscrew the valve
gradually allowing the pressure inside the bath rise to that of the atmosphere.
f. Remove and change the wax or embed the tissue as necessary.

NB – Never turn off the water pump while the stop-clock is still open. If this precaution is
not observed , water may be sucked back into the tap bottle and the vacuum chamber. When the
oven is not in use, the rubber washer should be removed. Prolonged exposure to high
temperatures causes the rubber to perish.
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 4. EMBEDDING / BLOCKING
It is also referred to as casting. After tissues have been infiltrated with molten paraffin wax
(M.P.W), it’s necessary to obtain a solid block of wax containing the tissue. This is done by filling
a mould with fresh molten paraffin wax (P.W.) and immersing in a piece of infiltrated tissue.
Orientate the tissue to ensure that it’s lying on the right plane. Finally allow the wax to solidify in
the mould. The solidification can be enhanced by immersing the wax block in cold water.

When the wax has completely solidified, it’s detached from the mould by hitting the mould gently
on the bench or by using a scapel knife. There are many types of mould available for use in the
laboratory.

They include following :-

1) Leukhard embedding mould

2) Metal trays moulds
3) Plastic ice cube trays.
4) Tissues – tek – rings.
5) Aluminum foil boxes
6) Water glass and glass tubings.

LEUKHARD EMBEDDING MOULD

The leukhard embedding moulds are made of rust-proof material e.g. Brass. They are supplied in
various lengths and are L-shaped. They make enclosures of different sizes. Their depth is usually
1.5 – 2.0 cm.

The L-shaped pieces are placed on a metal plate or glass which acts as a base of the mould. This
embedding moulds are convenient for use in a routine laboratory. To avoid wax sticking on he
mould or plate, it’s advisable to smear thinly glycerine on both the L-shaped pieces and the base
plate. The moulds are also cheap to buy. Blocks of wax produced by this method will require
wooden holders for microtomy.

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 ATTACHMENT

This is achieved by use of heat using a wooden handle spatula.

Procedure

i) The spatula is heated in a bunsen burner flame until it’s red hot.
ii) Melt the underside of the wax containing the tissue to be cut and allow the molten wax
to drop on the wooden holder.
iii) Quickly withdraw the spatula and press the wax block on the wooden holder sand-
witching a label that carries the identification number.
iv) Hold it there for a few seconds.
v) Reheat the spatula and seal the edges of the wax block on the wooden holder.
vi) Allow to cool.

NB – 1) The label should be written in a pencil because ink can be absorbed or dissolved
by the reagents during processing.
2) The label should be treated in molten paraffin wax so that it is not soaked with other
reagents i.e. becomes waterproof.
3) Ensure that the edges of the wax block are parallel.

TISSUE - TEK EMBEDDING MOULDS

These are supplied in various sizes and are quick to use. They also economical on molten paraffin
wax. The plastic part is used in conjunction with a stainless steel base or mould.

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The tissue to be embedded/blocked is first placed in the mould previously smeared thinly with
glycerine or sprayed with an aerosol supplied with the mould. The plastic ring is then placed in
position on the mould. The identification is penciled on the plastic ring. Molten paraffin wax is
then added until it brims the plastic ring.

Allow the wax to cool and when hardened, detach the mould from the wax block containing the
tissue in the plastic ring ready for cutting.

The plastic ring serves as block holder in the microtome chunk. After cutting the sections, the
blocks are stored in the rings.

Advantage

This method saves a lot of time but requires a lot of storage space. The moulds and the rings are
very expensive.
CELLOIDIN TISSUE EMBEDDING

Celloidin has rubbery consistency.

It is nitro-cellulose soluble in equal parts of alcohol and ether.
Thus, with celloidin embedding stage of clearing, dehydration and being transferred from
absolute. alcohol to thin celloidin finally in thick celloidin is avoided.
Tissues are cut and stored in dilute alcohol.
Advantages
1. Gives greater support to mixed tissues e.g. skin with subcutaneous fat.
2. Gives greater support to hard tissues e.g. bones.
3. Used when it is desired to avoid use of heat e.g. in central nervous
system.

GIANT GELATIN SECTIONS

Derived by Gough and Wentworth 1949.

Good to lung tissue.
After fixation whole lung tissue are embedded in gelatin.
Tissues-gelatin is allowed to set and then formolized.
Section of 300-400µm are cut on special large microtome.
Sections are then laid on sheet of perpex covered with filter paper and allowed to dry.
Tissues are adhere to the filter paper and can be filled in a book.

FROZEN TISSUE SECTIONS

Unfixed or fixed tissues are frozen with carbon dioxide or liquid nitrogen (cryo-
precipitants).
They are then sectioned in special microtome.
Advantages

Used when rapid diagnosis is required.

Used to demonstrate material soluble in alcohol or clearing agents.
NB: Friable materials are embedded in gelatin prior to cutting.
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 CRYOSTAT SECTIONS

Is a deep freeze type cabinet to house the microtome, knife and tissue and maintain them to a low
temperature (-10 to -20 °C) during the process of cutting.
Enables rapid sectioning of fresh unfixed tissues which have been quick frozen at a very low
temperature (-160°C).
Frozen sections are good for histochemical assessment and also for fluorescent antigen-antibody
technique.
Advantage
a) Rapid than freeze drying- thus used for rapid diagnosis
b) Present less technical problems than freeze dying .
Home work

i) In tissue sectioning what is meant by the following terms?

a) Ribbon
b) serial section ?

ii) Read on sectioning of Celloidin

Attachment of the wax block containing tissue to a wooden holder .

─Necessary for tissues which have been blocked by either Leukhard Embedding moulds , metal
ice
cube trays, watch glasses, glass tubings and aluminum foil.
─Not necessary for tissues which have been blocked using tissue - Tek method. Wooden holder
provides attachment on the microtome chuck.

Procedure

i) Using a hot spatula attach the label to the wooden block using wax
ii) Trim the paraffin wax block so that all sides are parallel, leaving an allowance of
minimum
1
/6 between the tissue and edges.
iii) Heat the spatula to near red hot and hold the tissue block above the wooden block.
iv) Quickly, melt some wax from the side further from the tissue and let the molten wax fall
on
the wooden block.
v) Quickly place the tissue block on the wooden block and hold it in position for a few
seconds.
vi) Heat he spatula again and seal the sides of the wax block to the wooden block completely
and allow the wax to solidify.
vii) Clean off any wax on the sides of the block ready for sectioning.

Section Cutting

In order to cut sections of good quality, four factors must be considered. These are
i) Good knowledge of the microtome in use
ii) Practical experience (skills)
iii) Tissues must have been properly fixed / processed.
iv) You must have a good and properly sharpened microtome knife.
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Paraffin wax embedded tissues should be cut rotary, rocker Microtomes and the recommended
section thickness is between 3-6m thick (average is 5m)

Trimming

It is necessary for the wax block of all wax blocks after Leukhards embedding mould.

i) A sharp scapel knife is used and

ii) A margin of 3-5 mm is allowed around the tissue.
iii) The edges must be parallel.
iv) The size of the wax block trimmed should be enough to fit on the wooden holder onto
which
it will be attached using a hot spatula.

Preliminary sectioning

Preliminary section cutting is required on all blocks whatever the mould used.

i) The block of wax containing the tissue is clamped on the microtome holder and
secured tightly ensuring parallelism with the knife.
ii) Preliminary sectioning is then carried out at 10 – 24µm thick until the whole surface of
the tissue is cut.
NB. i) The clamping of the tissue on the microtome must ensure proper position of cutting.
Tissues embedded in tissue – Tek are clamped onto the microtome chuck directly, the
support being derived from the plastic ring.
ii) It is also important to have a knife preserved for preliminary cutting and another for final
sectioning.

MICROTOMY (Final Sectioning)

 After preliminary cutting, turn the feed mechanism of the microtome backward to clear the
block from the knife. The knife is then removed and a suitable knife preserved for sectioning
is inserted and secured tightly into the microtome.
 The thickness gauge is then reset at 5m thick and the microtome operated to obtain a ribbon of
serial sections.
 The operation of the microtome involves a steady and rhythmic cutting of tissue.
 The ribbon of sections produced can be held between thumb and index finger and gently pulled
as the ribbon gets longer.
 When the ribbon is about 6 inch , separate it from the knife using a camel hair brush.
 The ribbons may be stored in a section box/book for later use.

NB – To obtain ribbons of serial sections, the knife must have been set at an angle of tilt between
0 – 5 degrees. Beyond both limits, it will be difficult to obtain ribbons.

Homework :- difficulties encountered during tissue sectioning.

State the -problem
-causes
-solutions.

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