Unit 1: The Microtomes

Introduction:
The earliest form of microtomy was the freehand sectioning of fresh or fixed
material using a sharp razor. The section produced, could, with practice, be
quite thin and translucent.

Modern microtomes are precision instruments designed to cut uniformly thin

sections of a variety of materials for detailed microscopic examination.

For light microscopy, the thickness of a section can vary between 1 and 10
microns (thin sections).

For electron microscopy, the thickness of a section is usually of the order of

10 nanometers (ultra-thin sections).

All microtomes consist of three main parts:

Base (microtome body)

Knife attachment and knife

Material or tissue holder

With most microtomes a section is cut by advancing the material holder
towards the knife whilst the knife is held rigidly in place. The cutting action
which can be either in a vertical or horizontal plane is coupled with the
advance mechanism so that the material holder is moved after each cut.
The distance moved is pre-selected using a scale setting on the microtome
body and usually extends between 0.5 and 50 microns on microtomes
cutting thin sections and from less than 60 nm to over 500 nm on
machines cutting ultra thin sections.

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Types of microtomes and their use:

1- ROTATORY MICROTOME:

Rotatory microtomes are general purpose microtomes for cutting semi-thin

to thin sections for light microscopy.

The microtome operation is based upon the rotatory action of a hand wheel
activating the advancement of a block towards a rigidly held knife. The block
moves up and down in a vertical plane in relation to the knife and therefore
cuts flat sections.

Available machines range from lightweight, rotatory microtomes suitable for

cutting paraffin wax embedded material in a continuous ribbon to heavy
duty, motor driven instruments used with a slow, continuous speed and
retracting advance movement to section hard material embedded in
synthetic resin. The rotatory microtome can also be found in most cryostats
for cutting frozen sections.

Section thickness settings range from 0.5µm to 60µm on most machines.

Sections of paraffin wax embedded tissues are normally cut within the range
3 to 5µm whilst resin sections are between 0.5 to 1µm.

Rotatory microtomes are especially suited to cutting sections using

disposable steel knives.

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2- SLEDGE MICROTOME:

These are designed for cutting large blocks of paraffin and resin
embedded material including whole organs, for light microscopy.
The knife holding clamps allow the knife to be offset to the direction of cut,
a major advantage when sectioning large, hard blocks. The microtome is
very heavy (for stability) and hence, is not usually subject to vibration.

3- ROCKING MICROTOME

Produced in large numbers early in

the 20th century, the rocking
microtome, comprising three
moving parts, is extremely
reliable. Designed only for
cutting paraffin sections the
tissue moves through an arc as it
advances towards the knife (the
slightly biconcave knife is used) which is held rigid causing the sections to
be cut in a curved plane. Very thin sections are difficult to obtain and
one major disadvantage is a limit to the size of block which can be
cut. Because of the lightness of the frame the microtome has a tendency to
move during cutting. The rocking microtome has largely been replaced by
the more precise rotatory microtome although it is re-appearing in portable
cryostats.

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4- FREEZING MICROTOME:

This form of microtome is used for cutting thin to semi-thin sections

of fresh, frozen tissue. The freezing microtome is equipped with a stage
upon which tissue can be quickly frozen using either liquid carbon dioxide,
from a cylinder, or a low temperature recirculating coolant. Some cooling
systems also allow the knife to be cooled at the same time. The cutting
action of the freezing microtome differs from those described previously as
in this case the knife is moved whilst the tissue block remains static. The
block moves by a pre-set amount, in microns, at the end of each cut.
Consistent, high quality, thin sections are very difficult to obtain
with this type of microtome.

5- CRYOSTAT

A cryostat is primarily used for cutting sections of frozen tissue. The

cryostat commonly consists of a microtome contained within a refrigerated
chamber, the temperature of which can be maintained at a preset level.

A recent innovation has the body of the microtome positioned outside the
refrigerated chamber. The cryostat usually contains a rotatory microtome
although some portable units utilize a rocking microtome. With the object,
object holder and knife all at the same temperature and all other conditions
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for cutting the material optimal, sections as thin as 1 micron are
possible.

Cryostat

6-VIBRATING MICROTOME
Originally conceived as a microtome which could produce high quality
sections of fresh, unfixed specimens. Principally, it is used to
prepare the tissue blocks for ultramicrotomy.

The name of the instrument derives from the high speed vibration produced
in a safety razor blade to provide the cutting power. The amplitude of
vibration is adjusted by altering electrical voltage applied to the 'knife'.
Different degrees of vibration are required to produce sections from varying
densities of material. To prevent tearing, soft material is cut whilst
immersed in a fluid which also aids in dissipating heat produced at the
vibrating edge of the razor as it cuts.

The principle of the Vibratome® is a vibrating blade intersecting the

specimen underneath the surface of a liquid bath which lubricates the cut.
There are five parameters under the control of the operator:

[Link] of the bath (usually a physiologically compatible buffer).

[Link] of the bath (the softer the specimen, the colder the bath
should be).
[Link] (the softer the specimen, the slower the speed).
[Link] (the softer the specimen, the higher the amplitude).
[Link] angle (the softer the specimen, the steeper the angle of attack
required).

One must, by trial and error, find the right mix of these parameter settings.

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7- ULTRAMICROTOME

The ultramicrotome is used to prepare ultrathin sections for light and

electron microscopy. Very small samples of tissue are usually embedded
in hard resin before cutting. It has been reported that sections can be cut as
thin as 10 nanometres.

Two forms of advance mechanism have been developed in this style of

microtome. The thermal mechanism relies upon heat induced expansion in a
bifurcated metal strip whereas in the mechanical form a microprocessor
coupled to a precise stepping motor controls the advance mechanism. The
cutting stroke is motor driven to provide a regular, smooth motion for
sections of even thickness and constant reproducibility. Knives are usually
made from glass, diamond or sapphire. The block is brought to the knife
edge under microscopical control and as each section is cut it is floated on
to a water bath adjacent to the knife edge.

6
Unit 2: The Microtome Knives
Types of microtome knives:
1- STEEL KNIVES
Steel microtome knives are manufactured from high quality carbon or tool grade
steel which is heat treated to harden the edge. The steel should be free from
impurities, contain anti-corrosives and be rust-resistant. The best knives are
those that are fully hardened. Those which are only surface hardened lose the
cutting edge very quickly once the hardened area is removed through repeated
re-sharpening.

a) NON-CORROSIVE KNIVES FOR CRYOSTATS

These are manufactured from hardened, heat treated stainless steel free from all
impurities and containing 12 to 15% chromium.

b) TUNGSTEN CARBIDE

Knives manufactured from high quality tungsten carbide are non corrosive,
practically non magnetic and 100 times harder than hardened tool steel.
The knives have excellent resistance to wear but are brittle because of their
extreme hardness and should be handled carefully. Up to 30,000 serial sections
of undecalcified bone embedded in methacrylate per sharpening has been
reported.
c) DISPOSABLE BLADES
Disposable microtome blades are essentially refined, thickened razor blades.
When held in a specially adapted knife holder the blades consistently produce
high quality sections and have replaced conventional microtome knives in many
instances. All disposable blades are manufactured from high quality stainless
steel although there are different grades according to the thickness of the blade.
The edge of disposable blades can be coated with platinum6 or chromium7 to
enhance strength and prolong cutting life. Teflon coated blades are particularly
suitable for use in cryostats as these offer reduced cutting resistance and
minimal friction. The smaller, thinner disposable blade also reaches cryostat
chamber temperature more rapidly than a conventional knife minimizing time
delay during blade exchanges or temperature adjustments.
Disposable blades need to be held rigid in a
special holder to prevent vibration during the
cutting stroke. These knives consistently
produce high quality sections virtually free
from compression.

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2- GLASS KNIVES
The cutting edge of glass knives used for conventional sectioning is parallel to
one surface of the glass ('Ralph knives' with edges of 25 or 38 mm) whilst in
those used for ultramicrotomy it is across the thickness of the glass. A
commercial glass knifemaker is recommended to ensure consistency and
reproducibility of the knife edge. Different profiles of 'Ralph knife' for cutting
sections from different embedding media can be produced very quickly.

Glass knife holders are available so that 'Ralph knives' can be used with a
rotatory microtome. Glass knives are hard but brittle and care is required
with their handling. These knives deteriorate with storage due to changes in
the 'flow' or 'strain' of the glass after fracture and from oxidation impurities
remaining in the hardened glass after manufacture. Knives should thus be
prepared immediately before use.

3- DIAMOND KNIVES
Diamond knives are manufactured from gem quality diamonds. Although this
makes them very expensive the knives are extremely durable, because of the
hardness factor of the diamond, and are used primarily for cutting ultrathin, resin
sections.

4- SAPPHIRE KNIVES
These knives are manufactured from one piece of solid sapphire artificially
produced from an alumina monocrystal under computer controlled thermal
conditions. Sapphire is harder than tungsten carbide or glass which ensures high
durability of the cutting edge for all types of material. The only restriction when
using a sapphire knife is block size as the knife edge is limited to 11 mm. A
special knife holder is required.

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Profiles of steel knives
Microtome knives of hardened steel are made to four different profiles for
cutting various materials.

Profile A: strongly plano-concave/biconcave

One surface of the plano-concave knife is
straight whilst the other is hollow ground.
The bi-concave knife has two hollow
ground surfaces. Both knives are
extremely sharp and are used for
cutting soft, celloidin embedded
material. These knives are not suitable for
relatively hard materials, which cause the edge to vibrate and produce the
phenomenon known as chattering. To obtain the best result the knife should
always be oblique to the object when cutting sections.

Profile B: plano concave

This knife is similar to a profile A knife
but has a thicker back. It is used for
cutting sections from material which is
too hard to cut with a profile A knife but
can also be used for softer materials embedded in paraffin wax.
This knife should be positioned obliquely to the material being sectioned.

Profile C: wedge Shaped

The wedge shaped knife has more rigidity
than profile A or B knives and can
therefore be used for cutting harder
materials. Because of the extra thick
nature of the wedge at the tip this type of
knife cannot be ground as sharp as profile A or B knives. Commonly used
for cutting sections from paraffin wax embedded material, frozen sections,
cryostat sections and for small, synthetic resin embedded material.
With this style of knife the cutting plane is transverse to the object.

Profile D: plane Shaped.

This knife will cut hard and tough
material as it has greater stability than
any of the other profile knives. As only
one bevel provides the cutting edge this
knife is the least sharp of all of the profiles. It is commonly used for cutting
synthetic resin blocks, hard materials embedded in paraffin wax, large wax
blocks and various substances used in industry.
The cutting edge of all steel knives is produced by grinding a bevel on each
side of the knife for profiles A, B and C, or onto the angled surface of a
profile D knife. The bevel faces enclose a sharper angle than the main
surfaces of the knife.
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Sharpening steel knives
A sharp knife edge free from imperfections is essential for the production of good
sections. However, with the introduction of disposable knives the practice of
sharpening traditional microtome knives has all but disappeared. Yet the need for a
more stable cutting edge occasionally arises and it becomes necessary to sharpen a
solid knife.
This can be achieved manually or by using an automatic knife sharpening machine.
Automatic machines tend to remove more metal during sharpening so that knives
become worn quickly. Manual methods on the other hand remove far less metal
but require more skill, experience and time to produce a satisfactory edge.
Fine honing and stropping is best done manually using diamond lapping
compounds. Coarse sharpening can be performed manually or by using automatic
knife sharpening machines.
COARSE KNIFE SHARPENING
Automatic
In most instances the knife is held rigid whilst being moved against a rotating
drum, rotating wheels or a lapping plate. Abrasive compounds of various grades,
from coarse to fine, move against the knife edge. The knife blade is automatically
turned at preset intervals so that each side is evenly sharpened.
Lubricants are essential for successful knife sharpening. These agents cool the
knife edge (heat generated by the abrasive procedure can destroy the 'temper' of
the steel) and continually remove the fine metal particles, produced by the
abrasive process, away from the knife edge (see later).
Manual
Coarse honing can also be performed manually using a lapping stick coated with
diamond paste containing industrial diamonds with sizes up to 25 µm diameter.
Lapping sticks made from hard woods are used for coarser honing whilst a soft
wood is better for finer honing and stropping.
FINE KNIFE SHARPENING
Fine honing is achieved by applying diamond paste, containing industrial
diamonds of 1 µm or less, to a lapping stick which is then moved against the
knife edge. For manual honing and stropping a knife bevel must be fitted to
the back of the knife to ensure the correct angle of bevel for the edge of the
knife.
STROPPING
Stropping polishes the knife edge and removes fine metal burrs retained
along the edge after honing. Leather, coated with a fine rouge powder or a
lapping stick coated with a diamond paste containing industrial diamonds of
less than 0.5 µm are effective for this purpose. It is important that very
little pressure is used when stropping.
MANUAL METHOD FOR COARSE OR FINE SHARPENING AND
STROPPING
Microtome knives are extremely sharp and must always be treated with due
care and particularly during manual sharpening. A simple distraction can
result in severe injury. The simplest manual sharpening method is that
using diamond paste. These contain diamonds of a known size and enable
10
the operator to remove minute quantities of metal and obtain a finish to a
knife edge which is almost impossible to achieve using any other form of
technology.
MATERIALS REQUIRED
1 Each knife must have its own stropping bevel which is essential to
maintain the correct cutting angle to the knife edge.
2 A holding device to hold the knife firmly during the sharpening procedure.
3 Diamond paste containing industrial diamonds of 1µm diameter for
stropping and finishing.
4 Diamond paste containing industrial diamonds of 6µm diameter for fine
sharpening.
5 Diamond paste containing industrial diamonds of 14µm diameter for
coarse sharpening.
6 Hardwood and softwood lapping sticks measuring 15 x 2 x 0.75 cm.
7 Aerosol can of lapping fluid.
METHOD
1 The stropping bevel is placed along the back edge of the knife to ensure
the correct bevel to the edge of a knife is produced.
2 The knife is clamped in position in a holding device firmly held by its
handle.
3 A small quantity of diamond paste is rubbed evenly over the surface of a
lapping stick. (During the sharpening procedure diamonds become
embedded in the wood of the lapping stick, particularly the softwood sticks
used for fine sharpening and stropping, so that in time paste need only be
applied sparingly.
4 After coating with diamond paste the lapping stick is firmly placed against
the side of the knife with the stick touching both the edge of the knife and
the stropping bevel.
5 The lapping stick is then moved along the knife in a filing action with
continuous, even pressure maintained throughout the stroke. Each side of
the knife must be given the same number of strokes. Fifteen strokes on
each side should be sufficient to sharpen a blunt but undamaged edge
whereas thirty strokes are required for stropping .
Other manual sharpening methods use natural stones, slabs of stone or
glass plates as a supporting surface for a lubricant and an abrasive
compound.
Lubricants are essential with these methods. It is best to use a non
aqueous lubricant (thin lubricating oil) in order to prevent the knife edge
from rusting. If an aqueous lubricant is used the knife should be thoroughly
cleaned after the sharpening and coated with a fine film of oil. Soap,
detergent, glycerol and water soluble oils have all been used as lubricants.
Abrasive powders various agents act as abrasives including carborundum
(silicon carbide), alumina (aluminium Oxide), magnesium oxide, chromium
oxide and iron oxide (rouge).
Abrasive powders are supplied in a variety of grade sizes. Coarse powders
are effective for restoring damaged edges whilst finer powders are used for
polishing and stropping.

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Unit 3: Section Cutting

PARAFFIN SECTIONING:
With a good knife edge and hard, well set and homogenous paraffin wax, sections of 1
µm are possible if the block face is no larger than 1 cm x 1 cm. In a block of this size
the tissue should occupy approximately 50% of the surface to be cut. However the
normal practice since block sizes can be much larger than this is to cut ribbons of
sections at 3 to 5 µm.

The objective is to produce a ribbon of artefact free, flat sections from which one to
several are selected and mounted on to clean slides.

METHOD

1. Set the blocks on to a cold surface to harden the face to be cut (a refrigerated cold
plate or ice). Avoid prolonged cooling and very cold surfaces as both can cracks the
surface of the paraffin wax block.

2. Install a sharp, trimming knife in the microtome and set the correct clearance angle,
normally 2 º to 5º.

3. Trim a paraffin wax block, with a sharp blade, so that the sides are parallel and 2 to 3
mm of wax surrounds the tissue.

4. Fit the trimmed block into the block holder and orientate so the edge offering least
resistance meets the knife edge first

5. Advance the block until it just touches the knife edge.

6. Coarse cut the block at 15 µm until the full face has been trimmed.

7. Return the trimmed block to the cold surface for 1 to 2 minutes.

8. Set the advance feed to the desired thickness (3 to 5 µm for most purposes).

9. Remove any debris associated with coarse cutting from the knife edge with alcohol.
(Xylene should not be used as it often leaves an oily remnant on the knife to which
cut sections will stick).
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10. Install a fresh, sharp knife in the microtome or move the previous knife to a new,
unused area.

11. Re-install the cold block in the microtome and cut a series (or ribbon) of sections at
the required thickness. Gently breathing upon the sections as each is cut dissipates
static electricity, flattens the section and facilitates movement of the ribbon down the
knife. Section compression is minimized by using a sharp knife set to the correct
clearance angle.

12. The ribbon is separated from the knife edge with a moist camel hair brush and pulled
across the surface of a warm water bath. (Section expansion will compensate for the
compression caused when cutting).

The temperature of the water should be approximately 10°C below the melting point of
the wax used in the block. Wrinkles in the section can be removed along with small
air bubbles trapped beneath the wax, by careful prodding with a moist camel haired
brush or metal probe (although the latter may damage to the section). Wrinkles
usually develop because different tissue components expand at different rates as
the section warms on the surface of the water.

13. Sections can be separated whilst floating on the water with gentle pressure from the
tips of forceps.

14. Sections are collected on to a clean glass slide. The slide is held vertical and mostly
beneath the surface of the water. The section is apposed to the slide which, when
lifted from the water, draws the section with it.

15. Slides are dried for a minimum of 10 minutes to ensure the section is firmly attached.
A hot plate set just above the melting point of the wax or a hot air oven are both
effective. For delicate tissues more prolonged drying in a hot air oven at a lower
temperature may prove beneficial. Overnight drying at 37°C is necessary for
maximum section adhesion. Slides left in the open for drying will accumulate dust.

All purpose glass slides are 76.2 x 25.4 mm (1" x 3"). Those preferred for light
microscopy are normally 1 to 1.2 mm in thickness and have ground and polished edges
to reduce the risk of injury. Slides with frosted ends are preferred as section/specimen
details can be inscribed with pencil rather than with a diamond stylus, which can create
small spicules of flying glass.
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Problems in section cutting:

1- Crumbling of sections
Cause: Inadequate impregnation (alcohol not completely removed)

Treatment: return to alcohol reprocess.

2- Scoring of sections with vertical lines:

Cause: Imperfect knife edge.

Treatment: Sharpen the knife

3- Alternatively thick and thin sections:

Cause: Imperfect knife edge.

Treatment: Sharpen the knife

4- Chattering Horizontal lines or furrows across the sections:

Cause: imperfect setting of the knife.

Treatment: reduce the tilt of the knife

5- Intermittent cutting (on and off cutting):

Cause: imperfect setting of the knife.

Treatment: Increase the tilt of the knife

6- Brittle and tough tissue:

Cause: Tilt of knife is too great

Treatment: decrease the tilt of the knife

7- Dirt in the sections:

Cause: dirt is present in the paraffin wax

Treatment: use clean wax

8- Calcium particles in the sections:

Use sledge microtome.

14
Factors affecting the production of good sections:
There are many factors which affect the production of good sections. Some of these are:

1) Fixation and embedding: Animal and human tissues are too soft when fresh to be
cut thinly. Some form of pre-treatment is required to harden the tissue to facilitate
cutting thin sections. This consists of either freezing or embedding tissues in a medium
which offers support for cutting.

2) Sharpness of the knife edge: A sharp unblemished knife edge is essential for
smooth, even sections.

3) The correct clearance angle:

Necessary to prevent compression
in cut sections. The correct
clearance angle is also important to
reduce friction as the knife edge
passes through the block. The
clearance angle is that between the
knife edge bevel and the block.
Various angles have been
recommended with between 2 and
4 degrees for paraffin sections and
between 5 and 7 degrees for resin
or frozen sections being most
effective. Determining the exact angle is largely a matter of trial and error.

4) Cutting angle: If the angle of bevel (cutting angle) is too great it can cause
compression in the cut section. If the angle is too fine the edge of the knife can vibrate
causing chatter in the section. A balance between these extremes will provide the best
results. Generally the sharper knife will have a finer cutting angle.

5) The hardness of the embedding compound: This reflects the thickness at which
sections can be cut. It is difficult to cut very thin sections from soft embedding
compounds. The following is a guide to the thickness at which sections can be obtained
from different embedding media ranging from soft (gelatin) to hard (resin):

Gelatin - 50 to 200 µm
Ice - 5 to 20 µm (frozen section)
Paraffin wax - 1 to 15 µm
Paraffin wax/resin mixtures - 0.5 to 2 µm
Resin - 0.05 to 1 µm

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Mounting Sections on glass slides:
Here are two ways of mounting:

1) Collect the sections in a warm water bath. When the sections are flat and expanded,
a clean glass slide is dipped obliquely into the water as close to the sections as
possible. Slowly withdraw the slide.

2) Place a clean glass slide on a hot plate and flood it with distilled water. Collect the
sections in this water. After being flattened, withdraw the water.

Drying the section:

Allow the sections on the slides to dry either in open air or in an oven.

Use of section adhesives:

Several adhesives are available. The most famous is the Glycerol-Albumin Mixture.

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Unit 5: FROZEN SECTIONS
Indications for frozen section

1) Rapid Diagnostic Purposes:

A) During surgical operations:
♦ when there is no preoperative diagnosis available.
♦ in case there was an unexpected intraoperative finding (e.g. peritoneal
carcinosis) must be clarified.
♦ Also, resection margins may be object of frozen sections.
B) During endoscopy or needle biopsy:
Small biopsies (obtained by endoscopy or needle biopsy) are rapidly
frozen sectioned to decide whether the tissue is representative enough for
further pathological diagnosis. These procedures may save the patient
from a second unpleasant endoscopy and is less expensive.
2) Several modern biological or molecular biological techniques require fresh tissue to
examine (blots, PCR and so on).
3) Tissue for bacteriology also requires fresh tissue.
4) Receptor analysis.
5) Immunohistochemistry.
6) Lipid demonstration.

Preparation of frozen sections

Fixation
Tissues for frozen sections may be fixed or unfixed. Unfixed tissues are
used in rapid, diagnosis and antibody works. Care should be taken to
minimize the risk of infection.
Fixatives commonly used include:
1) Formal saline which gives good results
2) Formal calcium used for histochemistry and lipid demonstration.
3) Formal ammonium bromide suited to demonstration of CNS
4) Acetic alcohol (Absolute alcohol 95 ml + acetic acid 5 ml) for rapid
cryosectioning.

17
 Cryoembedding

Here are some simple techniques which allow the user to embed tissues for frozen
section with a level of control and predictability that can surpass even paraffin
embedding. The cryoembedding medium used here is the OCT.

(1) Face down cryoembedding: This technique is similar to routine paraffin

embedding but instead of standing tissue into solidifying paraffin we are adhering
tissue flat to the base of a freezing well. This adhesive property of freezing steel
makes it easier to place tissues in precise position.

(2)Frozen block cryoembedding: Freezing tissue in a block of embedding

medium prior to slicing it transforms tiny specimens into large ones, flimsy soft
tissues into firm easy to cut specimens, straightens out rubbery curled tissues
and makes torn and perforated tissues whole again.

(3)Paper Embedding: Tissues placed in precise position on a small portion

of lens paper wetted with embedding medium are transferred on the paper to the
well floor. Allow precise flat embedding of the most delicate tissues or complex
arrangements of tissues.

Freezing
Four methods of freezing are in common use. Rapid cooling of the tissue is
required to prevent large crystals of ice forming which may cause distortion of
tissue structure. This phenomenon is called ice crystal artifact.

Methods of freezing are:

1) Liquid gases e.g. liquid nitrogen
2) Refrigeration devices: These are often present as an integral part of a
cryostat.
3) Thermoelectric modules
4) Aerosol sprays (containing dichloro-fluro-methane) which are seldom used.

The optimal temperature in the chamber is -18°C to -20°C.

The temperature on the chuck holder should be adjusted to reflect the type of
tissue being cut.

18
Sectioning:
The holder with tissue is then placed in the rack to the lower left of the cryostat box.
The tissue is trimmed; the anti roll placed in contact with the knife edge and section is cut
(8-10µ)
Section is picked up on a clean slide by lowering it onto the section.

Fixation:
Non fixed tissue is fixed before staining with the following Fixative. Slides are
placed directly in a coplin jar of the fixative which is kept at the room temperature
Acetic alcohol- fix for 1 minute
Absolute alcohol 95 ml
Acetic acid 5 ml
If there is delay in fixing the tissue there will be significant drying artifact

Staining

Rapid Haematoxylin and Eosin

1- Hydrate in 95°ro, 70% and distilled water, 5 to 10 seconds each.
2- Stain in modified Harris Haematoxylin for 1 minute
3- Wash in running tap water 5to 10 seconds
4- Blue in warm tap water 5 to 10 seconds
5- Counterstain in alcoholic eosin: 3dip
6- Wash in tap water
7- Dehydrate in 95% and absolute alcohol 5 to 10 seconds each
8- Clear in Xylol
9- Mount and coverslip.

Toludine Blue
1- Rinse fixed slides in tap water for 5 to 10 seconds
2- Flood with 1% aqueous toludine blue for 5 seconds.
3- Wash in tap water
4- Dehydrate, clear and mount in permount

19
Factors affecting the quality of frozen sections:

1. Cabinet temperature most cryostats are set to - 20°C however the optimum
temperature is probably about -10°C
2. Tissue temperature: The optimum temperature is -5°C and -10°C
3. Knife temperature: Temperature of knife between -10°C and -30°C
4. Maintenance of microtome (Defrosting, cleaning, drying and lubricating)
5. A sharp microtome Knife with a small facet gives best results
6. The anti roll plate or guide plate is a device attached to the front of the
microtome to ensure that sections, when cut lie flat along the knife and do
not curl up.
Maintenance and decontamination of the cryostat:

1. If applicable, ensure that the backup cryostat is functional

2. Turn the cryostat off, open the cabinet and allow defrosting.
3. Drain accumulated water.
4. Spray entire cabinet interior, microtome knife and freezing platforms with
disinfectant solution.
5. Close cabinet and all disinfectant to act for 15 minutes.
6. Rinse lightly with warm water, drain thoroughly
7. Rinse with 70% ethanol; allow acting for 5 minutes, drain
8. Rinse with 100% ethanol, rinse, drain
9. Rinse again with 100% ethanol, rinse, drain
10. Leave cabinet open until all areas are completely dry
1l. Lubricate according to manufacturers instructions
12. Turn power on and all cryostat to come to operating temperature
13. Check to ensure that all moving parts are freely moving. If any are frozen, work
with gentle pressure and additional appropriate lubricant until they are free. Add
small amount of additional lubricant once a part is moving.

20
Unit 6: Stains

Dye Structure and Colour

The components involved in histological staining are dyes and proteins. The
fundamental process involved is the chemical bonding between the carboxyl groups of
one and the amino groups of the other. The commonest bonds involved are ionic
bonds, although there are exceptions especially in the case of nuclear staining of DNA.

What dyes are?

In short, dyes are coloured, ionising, aromatic organic compounds.

Dyes may be toxic, carcinogenic or mutagenic, or harmful to your health in some other
way. Handle dyes with care! Put your own safety first!

Why dyes are coloured?

Colour in dyes is invariably explained as a consequence of the presence of a
Chromophore. Since, by definition, dyes are aromatic compounds their structure
includes aryl rings which have delocalized electron systems. These are responsible for
the absorption of electromagnetic radiation of varying wavelengths, depending on the
energy of the electron clouds.

Below are the usual chromophores seen in histological dyes:–

Azo-coupling (–N=N– Nitro-group (– Quinoid

) NO2) rings

Auxochrome:

The basic meaning of the word auxochrome is colour enhancer. This word was coined
because it was noted originally that the addition of ionizing groups resulted in a
deepening and intensifying of the colour of compounds.

Auxochromes are groups which attach to non ionizing compounds yet retain their ability
to ionize. While this definition is largely correct, it is also inadequate. This is because it
restricts the definition of the auxochrome to ionization, and does not comment on the
effect of auxochromes on the absorbance of the resulting compound.

Auxochromes are of two types. They may have a positive charge as the amino group
and its substituted variants. Or they may be negatively charged as the carboxyl and
hydroxyl groups, and the sulphonic group. This last is commonly used to convert basic

21
dyes to acid dyes. Both negatively charged and positively charged auxochromes may
be present on a single molecule.

Below are the usual auxochromes found in histological dyes:–

Amino group (– Aniline –

NH3) ring OH

Mordants:
Mordants are substances which combine the tissues and the stain, linking the two and
causing a staining reaction between them.
When mordants are necessary to perform staining, this called INDIRECT STAING. But,
if there is no need for mordants, this called DIRECT STAINING.
The mordants used in histology are:

Aluminu Iro
m n

Classification of stains
According to the source:

Synthetic
Natural

According to usage:

General (Routine) histological stains

Special stains

According to the their chemical activity:

Acidic stains
Basic stains
Neutral stains

22
 Staining Methods
Dyes may have microanatomical (histological) or cytological staining capacity:

1) Microanatomical stains: They used to demonstrate the general features of

tissues. But they do not emphysize on the detailed structure of the cells.
2) Cytological stains: They demonstrate the detailed structure of individual cells.

Vital staining: is the staining of living cells.

Specific Staining: These stain only particular constituents of cells and tissues, but
have no effect upon the remaining elements.

Negative staining: The target object to be examined remains unstained, while the
background is stained deeply. So, there will be a sharp contrast. It is used for the
examination of bacterial morphology.

Progressive stains: stains which colour the tissue elements in a definite order.

Regressive stains: those colour all the tissue elements at the same degree, and
necessitate washing out (differentiating) before the individual tissue elements can be
examined.

Indirect staining: It is the staining process that needs a mordant. E.g. Haematoxylin

Direct staining: It is the staining process that does not need a mordant.

Staining equipments:
There ere three methods of staining slides:

1) Using staining dishes.

2) Using staining racks.
3) Using Staining machines.

23
 Routine Histological Staining

Hematoxylin and Eosin (H&E) stain:

it is a good general stain for many types of tissue.

Solutions:
Gill's Hematoxylin

 Hematoxylin - 6 g
 Sodium Iodate - 0.6 g
 Aluminum Sulfate - 52.8 g
 Distilled Water - 690 ml
 Ethylene Glycol - 250 ml
 Glacial Acetic Acid - 60 ml

Eosin

 Eosin Y - 0.5 g
 Ethanol 96% - 100 ml
 Glacial Acetic Acid - 2 drops

Acid Ethanol

 4 ml of 25% HCl.
 100 ml of 70% Ethanol.

Procedure:
•Place slides containing paraffin sections in a slide holder (glass or metal)

•Deparaffinize and rehydrate sections:

3 x 3´ Xylene (blot excess xylene before going into ethanol)

3 x 3´ 100% ethanol

1 x 3´ 95% ethanol

1 x 3´ 80% ethanol

1 x 5´ deionized H2O

24
 •While sections are in water, skim surface of hematoxylin with a Kim wipe to
remove oxidized particles. Blot excess water from slide holder before going
into hematoxylin.

•Hematoxylin staining:

1 x 3´ Hematoxylin

Rinse with deionized water

1 x 5´ Tap water (to allow stain to develop)

Dip 8-12x (fast) Acid ethanol (to destain)

Rinse 2 x 1´ Tap water

Rinse 1 x 2´ Deionized water (can leave overnight at this stage)

•Blot excess water from slide holder before going into eosin.

•Eosin staining and dehydration:

1 x 30 sec Eosin (up to 45 seconds for an older batch of eosin)

3 x 5´ 95% ethanol

3 x 5´ 100% ethanol (blot excess ethanol before going into xylene)

3 x 15´ Xylene

•You can leave slides in xylene overnight to get good clearing of any water.

•Coverslip slides using Permount (or DPX) (xylene based).

•Place a drop of Permount (or DPX) on the slide using a glass rod, taking care to
leave no bubbles.

•Angle the coverslip and let fall gently onto the slide. Allow the Permount to spread
beneath the coverslip, covering all the tissue.

•Dry overnight in the hood.

Results:
Nuclei - blue
Basophilic cytoplasm - blue
Acidophilic cytoplasm - pink
Muscle tissue - pink
Connective tissue - pink

25
 Specific histological stains

These stains are classified into 3 categories:

1) Specific stains of connective tissue: they specifically stain the different

component of the connective tissue e.g. reticular fibers, collagen fibers & elastic fibers

2) Specific stains for particular tissue substances e.g. iron, mucin,

amyloid, etc.

3) Specific stains for micro-organisms e.g. bacteria & fungi.

I) The connective tissue stains

A) Collagen stains:
These include:

1) Masson's trichrome

2) Weigert-van Gieson stain

B) Reticulin stains:
These include:

1) Silver impregnation method

2) Van Gieson stain

C) Elastic fiber stains:

These include:

1) Verhoeff' stain: elastic fibers stain black, collgen red and nuclei blue

2) Weigert's Resorcin-fuchsin stain: elastic fibers stain dark blue to

black.

26
II) Stains for particular substances

A) Stains for Carohydrates:

E.g. Periodic acid Schiff (PAS) stain
B) Stains for Fat
E.g. Sudan stains
C) Stains for Amyloid
E.g. toludine blue and methyl violet
D) Stains for iron
E.g. Perl's Prussian Blue stain.

II) Stains for micro-organisms

E.g. Gram stain, Zeihl Neelsen stain

27
Unit 6: Immunocytochemistry
Definition:
Immunocytochemistry is the localization of antigens in tissue sections by the use of labeled
antibodies as specific reagents through antigen-antibody interactions that are visualized by
a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.

Since immunocytochemistry involves specific antigen-antibody reaction, it has apparent

advantage over traditionally used special and enzyme staining techniques that identify only
a limited number of proteins, enzymes and tissue structures. Therefore,
immunocytochemistry has become a crucial technique and widely used in many medical
research laboratories as well as clinical diagnostics.

There are numerous immunocytochemistry methods that may be used to localize antigens.
The selection of a suitable method should be based on parameters such as the type of
specimen under investigation and the degree of sensitivity required.

Tissue Processing

Fixation:

Tissue preparation is the cornerstone of immunocytochemistry. To ensure the preservation

of tissue architecture and cell morphology, prompt and adequate fixation is essential.
However, inappropriate or prolonged fixation may significantly diminish the antibody
binding capability.

There is no one universal fixative that is ideal for the demonstration of all antigens.
However, in general, many antigens can be successfully demonstrated in formalin-fixed
paraffin-embedded tissue sections.
The most common fixative solutions used for immunocytochemistry are the followings:

a) 4% paraformaldehyde in 0.1M phosphate buffer

b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer
c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate
buffer.
d) 4% paraformaldehyde with 0.05% glutaraldehyde (EM immunocytochemistry)

Some antigens will not survive even moderate amounts of aldehyde fixation. Under this
condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat
without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until
fixation with cold acetone or alcohol. After fixation, the sections can be processed using
standard immunohistochemical protocols.

Sectioning:

The largest proportion of material for immunocytochemistry is formalin-fixed, paraffin-

embedded. Paraffin sections produce satisfactory results for the demonstration of majority
of tissue antigens with the use of antigen retrieval techniques.

Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of
frozen sections still remains essential for the demonstration of many antigens. However,
the disadvantage of frozen sections includes poor morphology, poor resolution at higher
magnifications, special storage needed, limited retrospective studies and cutting difficulty
over paraffin sections.

28
Vibratome sections have some advantages when doing immunocytochemistry since the
tissue is not processed through organic solvents or high heat, which can destroy the
antigenicity. In addition, the morphology of tissue sections is not disrupted due to no
freezing and thawing needed. Vibratome sections are often used for floating
immunostaining, especially for pre-embedding EM immunocytochemistry. The disadvantage
of vibratome sections is that the sectioning process is slow and difficult with soft and poorly
fixed tissues. In addiction, the chatter marks or vibratome lines are often appeared in the
sections.

Whole Mount Preparation: Small blocks of tissue (less than 5 mm thick) can be
processed as whole mounts. The advantage of whole mount preparations is that the results
provide three dimensional information about the location of antigens without the need for
reconstruction from sections. However, the major limitation of using whole mounts is
antibody penetration may not be complete in the tissue, resulting in uneven staining or
false negative staining. So Triton X-100 or saponin treatments are used routinely for whole
mount immunocytochemistry to enhance penetration of the antibody.

Antigen Retrieval

The demonstration of many antigens can be significantly improved by the pretreatment

with the antigen retrieval reagents that break the protein cross-links formed by formalin
fixation and thereby uncover hidden antigenic sites. The techniques involved the application
of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in
an aqueous solution (commonly referred to as the retrieval solution). This is called heat
Induced epitope retrieval (HIER). Another method uses enzyme digestion and is called
proteolytic Induced epitope retrieval (PIER).

Microwave Oven, Pressure Cooker and Steamer are the most commonly used heating
methods. Other tools also include the use of autoclave and water bath. The heating length
of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20
minutes. Citrate buffer of pH6.0 is the most popularly used retrieval solution and is suitable
for most of antibody applications.

Improving antibody penetration is also important for immunohistochemical staining of

frozen and vibratome sections. Triton X-100 is by far the most popular detergent for
improving antibody penetration for immunocytochemistry. However, it is not appropriate
for the use of membrane antigens since triton X-100 destroy membranes. Some
researchers prefer the freeze and thaw method for the improvement of antibody
penetration.
Sodium borohydride (1% in phosphate buffer) treatment is also widely used to unmask
antigens, particularly in glutaraldehyde fixed tissue to reduce the glutaraldehyde linkages.

Blocking:

Background staining may be specific or non-specific. Inadequate or delayed fixation may

give rise to false positive results due to the passive uptake of serum protein and diffusion of
the antigen. Such false positives are common in the center of large tissue blocks or
throughout tissues in which fixation was delayed.

Antibodies, especially polycolonal antibodies, are sometimes contaminated with other

antibodies due to impure antigen used to immunize the host animal.

The main cause of non-specific background staining is non-immunological binding of the

specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue

29
sections. This form of background staining is usually uniform and can be reduced by
blocking those sites with normal serum.

Controls:

Special controls must be run in order to test the protocols and for the specificity of the
antibody being used.

Positive Control is to test for a protocol or procedure used. It will be ideal to use the tissue
of known positive as a control. If the positive control tissue showed negative staining, the
protocol and procedure need to be checked until a good positive staining is obtained.

Negative Control is to test for the specificity of the antibody involved. First, no staining
must be shown in the omission of the primary antibody or the replacement of the specific
primary antibody by an normal serum (must be the same species as primary antibody).
This control is easy to achieve and can be used routinely in immunohistochemical staining.

Direct Method:

Direct method is one step staining method, and involves a labeled antibody (i.e. FITC
conjugated antiserum) reacting directly with the antigen in tissue sections. This technique
utilizes only one antibody and the procedure is short and quick. However, it is insensitive
due to little signal amplification and rarely used since the introduction of indirect method.

Indirect Method:

Indirect method involves an unlabeled primary antibody (first layer) which reacts with
tissue antigen, and a labeled secondary antibody (second layer) reacts with primary
antibody (Note: The secondary antibody must be against the IgG of the animal species in
which the primary antibody has been raised). This method is more sensitive due to signal
amplification through several secondary antibody reactions with different antigenic sites on
the primary antibody. In addition, it is also economy since one labeled second layer
antibody can be used with many first layer antibodies (raised from the same animal
species) to different antigens.
The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine
or Texas red, and this is called indirect immunofluorescence method. The second layer
antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or
glucose oxidase, and this is called indirect immunoenzyme method.

PAP Method (peroxidase anti-peroxidase method):

PAP method is a further development of the indirect technique and it involves a third layer
which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable
peroxidase anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and
peroxidase, acts as a third layer antigen and becomes bound to the unconjugated goat anti-
rabbit gaba-globulin of the second layer. The sensitivity is about 100 to 1000 times higher
since the peroxidase molecule is not chemically conjugated to the anti IgG but
immunologically bound, and loses none of its enzyme activity. It also allows for much
higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies
and reducing non-specific background staining.

Avidin-Biotin Complex (ABC) Method:

ABC method is standard IHC method and one of widely used technique for
immunohistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase

30
or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin,
can be conjugated to a variety of biological molecules such as antibodies.

The technique involves three layers. The first layer is unlabeled primary antibody. The
second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin
peroxidase. The peroxidase is then developed by the DAB or other substrates to produce
different colorimetric end products.

31
Unit 8: Mounting Media

Introduction:
The final stage in the preparation of tissues for microscopy is mounting.

Mountants with a refractive index (RI) as close as possible to that of the tissue (usually taken as
the RI of fixed protein, between 1.53 and 1.54) will effectively render a section transparent.
With such specimens the only features visible will be those coloured by the staining method
used. A medium with a much lower or higher RI than that of the tissue will increase visibility
but is beneficial only if an overview of the specimen is required as the resolution can be poor
(evident in the extreme if sections are cleared and examined dry; the mountant in this case, air,
has a RI of 1.000). In some forms of microscopy (such as phase contrast) the use of a mountant
with an RI only slightly different from that of tissue is recommended to supplement contrast
enhancement.

To be effective, a mountant should possess certain characteristics, These

include the following:

 it should be colourless and transparent

 it should be able to completely permeate and fill tissue interstices
 it should have no adverse effect on tissue components
 it should be resistant to contamination (particularly microorganism growth)
 it should protect the section from physical damage and chemical activity (oxidation
and changes in pH)
 it should be completely miscible with dehydrant or clearing agent
 it should set without crystallising, cracking or shrinking (or otherwise deform the
material being mounted) and not react with or induce fading in stains and reaction
products (including those from enzyme histochemical, hybridisation and
immunohistochemical procedures)
 finally, once set, the mountant should remain stable (in terms of the features listed
above).

Mounted sections are often stored for many years and the use of an appropriate mountant is
critical to avoid deterioration in the specimen.

Types of mountants:
Mounting media are either hydrophobic or hydrophilic.
Hydrophobic mountants generally call for sections to be dehydrated (usually through a series of
graded ethanol solutions) and cleared (in a solvent miscible with the mountant) before the
mounting medium is applied. Sections are mounted in hydrophilic media directly from water.
Within each group, mountants can also be classified as adhesives or non-adhesives. In
general, adhesives harden through solvent evaporation and thereby fix the accompanying
coverslip to the slide.

32
Hydrophobic Mountants

The RI of hydrophobic (adhesive) mountants usually approximates that of tissue proteins

(fixed), and they provide firm adhesion of the coverslip, these mountants are the type most
frequently used.

1) CANADA BALSAM

This is an oleoresin obtained from the bark of the fir Abies balsamea (of the family Pinaceae),
native to North America. The dried resin is freely soluble in xylene and other organic solvents.
Originally introduced in about 1832 and widely used until only recently, Canada balsam has a
number of disadvantages: it yellows with age; is very slow to harden and; as it becomes
increasingly acidic over time, cationic dyes are poorly preserved and the Prussian blue product
of Perls' reaction is bleached.

REAGENTS REQUIRED
1) Canada balsam 55-65 g
2) Xylene 100 ml

METHOD
Prepare using the quantities indicated (greater amounts of resin will result in more viscous
solutions).

RI (solution) 1.523.

2) DPX (DISTRENE, PLASTICISER, XYLENE)

One of the most commonly used mountants, DPX is a colourless, neutral medium in which
most standard stains are well preserved. It is prepared by dissolving the common plastic,
polystyrene, in a suitable hydrocarbon solvent (usually xylene). A major disadvantage of
polystyrene media, however is that they set quickly and in doing so often retract from the edge
of the coverslip. This can be prevented by adding a plasticiser which is thought to resist the
effect by forming a mesh with the polymerised plastic.

REAGENTS REQUIRED
1 Polystyrene (Distrene 80) 18 g
2 Dibutyl phthalate 7.5 ml
3 Xylene 52.5 ml

METHOD
Prepare using the quantities indicated.

RI (solution) 1.523.

33
3) PERMOUNT

Application:

Permount is a toluene-based synthetic resin mounting medium. The right choice for both rapid
mounting and long term storage of slides. Its low viscosity allows for a thinner mounting layer
offering better optical quality and bubble-free preparations. Permount is clear with permanent
adhering qualities. Absolutely neutral. Will not become acidic or discolor with age.
It has a refractive index near that of a fixed protein which helps to keep images free of
distortion. Ideal for mounting coverslips to slides with thick or thin specimens Permount
preserves most biological stains with little or no fading when the slides are stored in darkness. It
contains an anti-oxidant to prevent the formation of annular rings and its high softening point
(155°C/311°F) makes it suitable for microprojection.

Notes:

Before using this product, open the adjustable tip to achieve the desired optimal flow rate of the
mounting medium. Place the bottle of Permount upside down in a cup or waste bottle.

4) EUPARAL

Euparal is a mixture of eucalyptol, sandarac (a resin from the tree, Tetraclinin articulata grown
in North West Africa), paraldehyde and camsal (camphor and phenyl salicylate). Its relatively
low RI (which is usually given as 1.483 but ranges from 1.478 at 20°C to 1.535 when solid)
makes it useful for mounting unstained sections. Another advantage is that slides may be
transferred directly from 95% alcohol eliminating the need for complete dehydration and
clearing. Some fading may occur in haematoxylin stained sections; in this situation the green (or
'vert') copper-containing form of Euparal is advocated.

5) RESIN

Sections of tissue embedded in plastic compounds (such as epoxy resins) can be successfully
mounted in liquid resin of the same type. Sections should be completely dry before applying
mountant which is best set using the same conditions prescribed for tissue blocks.

6) PHOTOSENSITIVE RESINS

Light polymerising resins have the advantage of very short setting times, requiring in the order
of 10-30 seconds exposure to UV light to harden completely. Once cured, however, the
mountant cannot be dissolved nor the coverslip removed (as might be necessary for restaining).
Acrylic based light sensitive resins are also suitable for fluorescence microscopy.

34
Hydrophilic (aqueous) Mountants

Aqueous (Hydrophilic) media are much used for procedures in which dehydrants and
hydrocarbon type clearants must be avoided. This will mostly relate to methods for
demonstrating lipid, enzyme identification, immunohistochemistry and fluorescent
microscopy.

Disadvantages of hydrophilic mountants are that they have relatively low RI, may induce stains
to leach from the section and many, being non adhesive, remain soft such that the edge of the
coverslip must be sealed to prevent drying out. Suitable agents (ringing media) for this purpose
are nail varnish, resinous mountants or paraffin wax.

Once made (or purchased), aqueous mounting media should be stored in small (10-30 ml)
screw-capped bottles or vials made of transparent glass or plastic. The cap can get cemented in
place, but is usually removable by soaking in warm water. If the medium becomes cloudy or
shows other signs of bacterial or fungal growth, throw it out and open another bottle.

1) WATER

Although of low RI (1.333), water serves as a convenient temporary mountant for some whole
specimens for examining certain microorganisms live (saline mount) and particularly when
checking sections during staining procedures.

2) GLYCEROL

Glycerol is also a useful temporary mountant but with a higher RI (1.460) and longer drying
time than water and glycerol may be added to other agents to retard drying and cracking.
Ringing the coverslip with a hydrophobic seal will extend the life of mounted sections, although
cationic dyes will diffuse into the medium over time.

3) Phosphate Buffered Glycerol (RI 1.47)

It is commonly used to mount sections for immunofluorescence.

REAGENTS REQUIRED
1 0.2 mol/l phosphate buffer 1 part
2 Glycerol 9 parts

METHOD
Prepare using the quantities indicated.

4) Buffered glycerol with anti-fade:

Buffered glycerol is used mainly for fluorescent immunohistochemical preparations. The high
pH provides for optimally efficient fluorescence of the commonly used labels. The added p-
phenylenediamine (PPD) or n-propyl gallate, retards fading.

Buffer:
35
Either 0.1M Phosphate buffer (pH 7.4): 10 ml
or 0.1M TRIS buffer (pH 9.0): 10 ml
Anti-fading agent:
Either p-Phenylenediamine hydrochloride: 100 mg
or n-propyl gallate: 500 mg
Glycerol: 90 ml

Keeps for at least 3 months, probably much longer, in darkness (which protects the anti-fade
agent) at -20°C. The working bottle is kept at 4°C, for a week or two.

This does not solidify, but the coverslip can be held in position by applying a little nail varnish
to its edges.

5) Glycerol jelly:

This traditional mounting medium is the most difficult one to use. If you can make a decent
preparation in glycerol jelly, you'll be able to use any other aqueous mountant with your eyes
shut.

Gelatin powder: 10 g
Water: 60 ml
Dissolve by warming and add:
Glycerol: 70 ml

Add either one drop of saturated aqueous solution of phenol ("liquid phenol") or 15 mg of
sodium merthiolate as an antibacterial agent. This can be kept for a few weeks at 4°C. Discard
when it becomes turbid or mouldy.

Glycerol jelly must be warmed to about 40°C to melt the gel before using. It commonly needs to
be freed of air bubbles, too. This can be done by warming the bottle (with cap loosened) in a
vacuum-embedding chamber.

After coverslipping with glycerol jelly, leave the slide on a warm (40-45°C), flat surface for
about 30 minutes, to let the medium soak into the section, and then remove it to a cool place to
set. It is quite difficult to make a bubble-free preparation with this medium, and the bubbles can
be tiny and numerous. Check with a microscope while the slide is still warm. If it's no good,
remove the coverslip by soaking in warm water, and try again.

6) Fructose syrup:

This medium is sticky enough to hold a coverslip in position. It is the easiest aqueous medium to
use, but too acid (pH about 4.5) for preserving basic dyes. Probably it could be made up in a
neutral buffer; I have never tried that myself. Fructose syrup is useful when you know you will
be removing the coverslip to do something else to the section.

Fructose (=levulose): 15 g

Water: 5 ml

Put together in a securely capped bottle and leave in an oven (about 60C) for 1-2 days, until all
the sugar has dissolved to form a clear syrup.

36
Keep for several months on the shelf at room temperature.

With long storage this medium will dry out and crystallize if the edges of the coverslip are not
sealed.

7) Apathy's gum-syrup:

This is another traditional mountant: troublesome to make, but easy to use. The thymol is to
retard the growth of microorganisms. Another disinfectant (see under glycerol jelly) could be
used instead.

Gum arabic (=gum acacia): 50 g

Sucrose: 50 g
Water: 50 ml
Thymol: One small crystal

Dissolve the ingredients, with frequent stirring and occasional heating on a water-bath. If big
lumps form, the gum may take several days to dissolve. The final volume should be
approximately 100 ml. Keeps for a few months at room temperature. Discard if it becomes
infected or if the sugar crystallizes.

The refractive index of Apathy's medium (about 1.5) is higher than that of glycerol jelly or
fructose, and it provides preparations that are almost as transparent as those made with a
resinous mounting medium.

8) Polyvinylpyrrolidone (PVP) medium

The composition can be varied according to individual needs. For immunofluorescence, it is

made up in a buffer and an anti-fading agent is added.

Polyvinylpyrrolidone (M.W. 10,000): 25 g

Water (or a 0.1 M phosphate or
TRIS buffer, pH 7.4 or 9): 25 ml
Dissolve the PVP by leaving for several hours on a magnetic stirrer. Then add:
Glycerol: 1.0 ml
Thymol: One small crystal

Bottles of PVP medium usually keep for 2 to 3 years at room temperature. Keep it in a dark
place if an anti-fading agent has been added. Discard if it looks infected or becomes too viscous.

This mounting medium is more runny than glycerol jelly or Apathy's. It is very easy to apply,
and not prone to bubbles. The refractive index is 1.46, but increases as the water evaporates at
the edges of the coverslip until unstained structures are barely visible. If you want a high degree
of transparency, wait several days before sealing the edges of the coverslip.

37
 RECEIVING AND PROCESSING
OF SURGICAL SPECIMENS

I. RECEIPT OF SPECIMENS

Hospitals vary with the way in which specimens are collected from the wards; but the
following requirements must be fulfilled:

1) The specimen containers must be robust and leak-proof.

2) Special collecting trays or boxes must be used and must be leak proof and able to
withstand repeated autoclaving or disinfection

3) All specimens must be carried upright.

4) The trays or boxes must be sterilised weekly or after any visible leak or spillage

5) Requisition forms should be kept separate from specimens to prevent them from
becoming contaminated.

6) In most routine histopathology departments specimens are received in a container of

fixative.

7) The volume of fixative should be approximately 10 times that of the specimen.

Unfixed specimens coming to the lab for frozen sections.

Some specimens are not placed in a fixative before sending to the lab, such as specimens
for histochemistry ( muscle biopsies), or specimens in which cytological imprints are
required e.g. Lymph node. The specimen is fixed there after

II. SPECIMEN IDENTIFICATION

Once specimens have arrived in the laboratory, they should be identified as follows

(1) Patients full name and date of birth

(ii) Source of specimen. The hospital ward from which the specimen originated must
be present on the specimen jar and the request form. The name of the hospital
doctor must also be noted.

(iii) Hospital number. The hospital number or unit number is a useful means of
identification and it is essential if the hospital uses a central computing system.

(iv) Date of the specimen is taken.

(v) Type of specimen, e.g. liver biopsy, skin etc.

(vi) Clinical history. Details of the patients clinical history including sex and notes of any
treatment such as drugs or radiotherapy.

38
Specimen details may be written into a day book or typed into a computer. Specimen is
then given a number. This number must be used for all subsequent procedures such as
labeling of cassettes or slides.

III. PROCESSING SURGICAL SPECIMENS

Every specimen should be examined before it is dissected and its microscopically

description carefully noted. The description should include :
(i) Accurate measurement of the. specimen with its weight

(ii) Photography of the specimen before dissection where it is indicated

The specimens should be processed according to the doctors request and the
clinical history of the patient.
Appropriate fixatives should be used . Extreme care should be taken to read the request
form and understand what is required to be done.
Following this the specimen is processed according to the standard methods laid do wn in
the laboratory manual.

RECORDS, STORAGE AND RETRIEVAL

The final reports produced by a Laboratory should ideally be stored in such a way that
they can be retrieved by a variety of means such as:

1. Name and date of birth

2. Hospital number

3. Accession Laboratory number

4. Diagnosis (usually in a coded form)

The retrieval systems commonly considered are:

l. Daybook manual filing system

2. Microfiche system

3. Computer system.

All of these data storage systems make use of the same information but they handle the
recording, storage, and retrieval of data with varying degrees of efficiency.

The daybook system being slowest and the computer system the fastest

I. Daybook/ manual filing system .

This is the oldest and probably still the most widespread method. Information of a
request form is entered into a daybook which is usually divided into the Accession
Laboratory Number, patients name, hospital ward doctor & type of specimen.

39
The disadvantages of the system are:

1. The time taken to store and retrieve information.

2. The large amount of space used for the storage of reports.

3. The inability to retrieve incomplete information.

The advantages of the system are:

l. Very little outlay for the equipment

2. No equipment maintenance

3. No problems with equipment breakdown

II. Microfiche Microfilm System

The recording of information from the request form like that of the daybook still applies.
However, the microfiche./microfilm system offers advantages in the storage and retrieval
aspects of the data handling.

Microfiche /microfilm can be divided into two operating system:

(a) An archival system is simply a mean of saving the space that paper reports use.

(b) An active system is similar to the archival system in that it saves floor space. Its main
advantage is its use for an automated filing and search facility.

III. Computers

A computerized date storage and retrieval system obviates the need for a daybook. The
computers are the best because they can store a lot of information in a very small area

The only major disadvantage of the system in the high costs

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