Unit IV G.

HONING

1. UNIT 4

1. Objectives:
Purpose of honing is to remove nicks and irregularity from the knife edge and to get a
sharp edge knife.

Honing is the process in which all the nicks and irregularities in the cutting edge of the
knife are removed to make the cutting edge straight and sharp. After prolonged use or
after cutting very hard tissue, the cutting edge will have become so damaged that
stropping will not resharpen it; small pieces of metal may have been removed which will
give a jagged edge, causing lines or tears in section cutting. A straight cutting edge and
the correct bevel must be restored by grinding the knife on a hone.

Types of honing:
a. manual method
b. automatic method

Types of hones:
1. Belgian black vein / Belgian yellow. Best for manual honing of knife.
It’s a natural stand stone Belgian block (yellow) with a black stone back.
2. Arkansas is a hard pale yellow white stone.
3. Carborundum is an artificial stone having coarse surface ,very useful for badly
nicked knives
4. Plate glass. Size ¼ inch to 3/8 inch thickness, length 14 inch and 2 inch width. It
is used with abrasive powders, it is cheaper, most popular, readily available and
after the use easy to clean.

Grades of honing material:

1. coarse
2. fine
Coarse particles are used for quick sharpening of badly nicked or damaged knives.
Coarse particles may cause nicks in the knife.

Taking care of a hone –

a. The hone is to be kept covered when not in use to prevent dirt and
grit from gaining access to the surface.
b. It is to be wrapped in soft cloth and stored in a shallow wooden box
with a lid.
Lubricants:
Lubricants are used in sharpening knives. Lubricants acts as coolant and protects the
temper of extreme edge.
Lubricants allow the flow away of fine metal particles from the knife edge and
movement of fresh abrasive particles towards the knife edge
 Lubricants reduce the tendency of stones pores to become blocked with finely
divided metal particles
Types of lubricants – 2 types
1. Aqueous lubricants
A. Liquid soap (1%), liquid detergent (10%)
B. Glycerol (50%)
C. Soluble oil (10-30%)
2. Non aqueous lubricants
a. Dilap fluid
b. Oils thinned with paraffin oil
Liquid paraffin is recommended because of high viscosity.

Abrasive powders:
Types:
1. Diamond- ideal abrasive available as paste or aerosol with particle size of 0.225
micron to 50 microns.
Advantage – particles retain their outline for more periods so that these can be
used for longer periods.
Copper, bronze and glass plates used in automatic sharpeners are superficially
impregnated with diamond particles.
2. Carborundum (silicon carbide) - available in 100 micron to 5 micron size
particles, used with light oil for both coarse grinding and polishing of knives.
3. Aluminum oxide (alumina)- size of particles is wide range up to 0.1 micron
.particles are not much hard and they are good for both sharpening and polishing
of knives.
4. Iron oxide – particles are soft and fine size can be used with aqueous and non
aqueous lubricants. Suitable for polishing the knife edge.
5. Ceric oxide – has got similar properties as that of iron oxide but more expensive.
6. Chromium oxide – particles are softer than alumina and are used on the softer
metal plates with aqueous or non aqueous lubricants. After the use hone must be
washed with warm soapy water to remove all metal particles from it, then
thoroughly rinse with water and dry it.

Method of honing:
1. keep the hone on a clean non skid surface
2. Small quantity of lubricants oil is applied and smeared on the surface.
3. Knife with handle and back sheath, cutting edge facing away from operator, is
placed heel near the center of the nearest end of the hone.
4. Hold the knife at either ends between thumb and fore finger
5. Place the knife at one end of the hone and push diagonally forward with the
cutting edge leading. This is called head to toe motion.
6. when the knife reaches the other end of the hone, it is pushed over on its back
and pulled back steadily towards the operator
7. The process is repeated 10 – 20 strokes or until the ragged edges are removed.
8. Put the knife in its box.
Precautions taken during honing –
1. Lubricant must be used.
2. Blade must be kept perfectly flat while honing because should the knife edge be
raised even the slightest, the edge will be turned.
3. After the honing is complete, the knife should be wiped with a piece of soft cloth
moistened with xylene.
4. The edge maybe then viewed with low power objective microscope to ascertain
the removal of nicks.

Regrinding of Knife:
When the cutting edge of the knife is damaged by large cricks which extend
below the actual cutting edge, regrinding has to be done. This is usually done by the
manufacturer.

Semi – automatic and automatic hones:

1. Semiautomatic hones - They are time saving, easy to manipulate. One big
disadvantage of this type of honing is that the manual feeding of the knife
across the revolving wheel cuss uneven pressure and variation in the rate
of honing resulting in uneven knife edge.
2. Automatic hones – Knife is fitted to holder and attached to main spindle
and allows the cutting edge to be in contact with the circular plate made of
metal or glass. The knife automatically turns over from edge to edge at
suitable intervals.

Plastic blades / disposable blades –

These can be adapted to fit most types of microtomes and necessity of sharpening
is eliminated. They are expensive but now – a – days commonly used.

Different types of microtome knife sharpeners.

1. Rotating wheel – made of glass or cast iron wheel, the knife is being drawn across
the periphery of the wheel.
2. Belt type – endless belt of leather, dressed with abrasive powders and driven by
electric motor. The knife is passed across the belt at right angles to the direction
of travel.
3. Rotating glass – a circular glass plate, horizontally mounted, smeared with an
abrasive suspension, driven by a constant speed by an electric motor, knife rests
freely against the surface of the moving plate. The knife moves forwards and
backwards across the surface of the plate and at intervals the knife is lifted and
turned over automatically.

IV h. Stropping:

Definition: Stropping is the process of polishing the already fairly sharpened edge after
honing.
 Stropping removes the buffs formed during honing. The knife edge cannot be
sufficiently sharp to cut good sections directly from honing or sharpening. Stropping
must follow sharpening. This is the final technique for sharpening the knife before cutting
sections.

Types of strops:
1. Rigid / fixed – Rigid type is preferred by many histotechnologists as it is easy to
manipulate and rounding of the knife edges is minimized.
2. Flexible / hanging
Strop is made from rump of horse and is fixed on wooden piece with handle of the
size of Length - 12 inch, Breadth - 2 inch, Height - 2 inch.
Back of strop is made of canvass to give support to leather during stropping.
Small quantity of vegetable oil is applied beneath the leather to keep the strops soft.

Technique:
1. Pullout hanging strop.
2. Put little mineral oil on the under surface of the strop.
3. Preliminary stropping is done on the canvass side.
4. Lay knife with its back obliquely on the strop, draw toe to heel with the cutting
edge facing the operator. The movement is that of “toe to heel”.
5. The action is opposite of honing.
6. Turn the knife on its back and brought back.
7. About 30 strokes are required, excess strokes may spoil the knife edge.
8. Before and after use, strop must be wiped (cleaned) with a soft cloth to remove
all particles.
9. Examine the knife edge under the microscope.
Automatic knife sharpener
1. It provides precise efficient and safe method of honing knives.
2. Knife is locked in knife holder; it turns the knife automatically for sharpening on
both sides.
3. Knife is sharpened on glass plate smeared with coarse abrasive to remove nicks
and later fine abrasive to polish the cutting edge of the knife.
4 abrasives are particles of aluminum oxide, magnesium oxide, chromium oxide,
carborundum.
5 abrasives are suspension of particles from 0.05mm to 0.1 mm

Care of microtome knife:

1. Store the knife in its box when it is not in use.
2. Clean the knife with xylol before and after use.
3. If it is to be stored for a long time, smear it with light weight oil to prevent
rusting.
4. Don’t keep knife flat on the surface.
5. use the back while sharpening
6. Use separate knife for cutting hard tissues like undecalcified bone and teeth.

Pearls: Some workers consider stropping unnecessary when honing has been done
properly; however stropping a knife after honing or after each session of section cutting
definitely enhances the cutting ability of the knife.

Summary:
Honing is the process in which all the nicks and irregularities in the cutting edge
of the knife are removed to make the cutting edge straight and sharp. Types of honing are
manual method, automatic method. Types of hones are Belgian black vein / Belgian
yellow, Arkansas, Carborundum, and Plate glass. Grades of honing material are
coarse and fine.
Lubricants are used in sharpening knives. Lubricants allow the flow away of
fine metal particles from the knife edge and movement of fresh abrasive particles towards
the knife edge. Types of lubricants – 2 types - Aqueous lubricants, and Non aqueous
lubricants. When the cutting edge of the knife is damaged by large cricks which extend
below the actual cutting edge, regrinding has to be done.
Different types of microtome knife sharpeners are Rotating wheel, Belt type, and
Rotating glass.
Stropping is the process of polishing the already fairly sharpened edge after
honing. Stropping must follow sharpening. This is the final technique for sharpening the
knife before cutting sections. Types of strops are 1. Rigid / fixed and 2. Flexible /
hanging

Key words:
Honing, hones, abrasives, Stropping, microtome, lubricants, Knife, automatic,

Self assessment:
 1. What is honing?
2. What are the types of hones used in histopathology lab?
3. What are the grades of honing?
4. What are the types of abrasives used in honing of the knife?
5. What are the precautions taken to protect the handling and use of the knife?
6. What are the lubricants used for honing of knife?

Answers:
1. Honing is the process in which all the nicks and irregularities in the cutting edge
of the knife are removed to make the cutting edge straight and sharp.
2. Types of hones are Belgian black vein / Belgian yellow, Arkansas, Carborundum,
and Plate glass.
3. Grades of honing material are coarse and fine.
4. Diamond, Carborundum (silicon carbide), Aluminum oxide (alumina), Ceric
oxide and Chromium oxide.
5. Care of microtome knife: Store the knife in its box when it is not in use. Clean
the knife with xylol before and after use. If it is to be stored for a long time, smear
it with light weight oil to prevent rusting. Don’t keep knife flat on the surface.
Use the back while sharpening. Use separate knife for cutting hard tissues like
undecalcified bone and teeth.
6. Types of lubricants – 2 types - Aqueous lubricants, and Non aqueous lubricants.

Further readings:
1. Text book of Histo Pathological techniques by C.A.F.Culling.
2. Histological Techniques – A Practical Manual by K.Lakshminarayanan.
 IV i. Section cutting

Introduction:
Clinical diagnosis need to be confirmed by laboratory investigations for a proper
patient management and therapy. For Histopathological diagnosis, a good thin tissue
section is very important. Well fixed paraffin embedded block are pre requisites for
getting thin sections.

Objectives:
To get thin good quality tissue section for staining by histotechnologist and
histopathological examination by the histopathologist.

Instruments required:
1. Good microtone
2. Sharp microtone knife
3. Properly processed tissue
4. Block holders
5. Water bath
6. Slide warmer
7. Forceps
8. Number 0 brush
9. Albuminized slides
10. Experience

Once embedded, tissues are cut into thin sections ready to be placed on a slide. This is
done with a microtome, an apparatus for feeding the blocks past an ultrasharp blade with
micron level precision.

Trimming the paraffin block

When everything has gone well in fixing, dehydrating, infiltrating the specimen and
embedding it, you should have obtained a paraffin block containing the specimen
surrounded with –generally- a too generous amount of paraffin wax. The surplus paraffin
needs to be removed and the paraffin block trimmed in its final form prior to sectioning.

When several pieces of specimen are embedded in one block (as in the picture below)
and you only want to section one of them it’s necessary to cut and trim the block too.
Paraffin block containing both pathological and normal rabbit liver tissue, embedded in
paraffin wax using a matchbox.

How thick the paraffin layer surrounding the specimen should be is dictated by the final
slide one has in mind and by personal preference of the microtomist. As a general rule,
it’s a good practice to make the paraffin layer surrounding the specimen not too thick: the
larger the surface of the paraffin block to be sectioned, the higher the risk of problems in
section cutting and it serves no purpose to cut blank paraffin. Cutting only makes your
microtome knives/blades dull at the end, so it’s not a bad idea to economize on knife
sharpening/disposable blades and to use the knife/blade only for sectioning what really
matters: the embedded specimen. On the other hand: there should be enough paraffin left
to handle the sections in a comfortable manner.

Especially when you want to mount several serial sections under a common cover slip the
surrounding paraffin should be kept to a minimum: 1 or 2 mm. The thinner the
surrounding paraffin coat, the more sections can be mounted under a single cover slip.
When only individual sections are to be mounted onto slides the surrounding paraffin
coat can be kept larger so that the sections can easily be manipulated with a pair of fine
tweezers or a camel hair brush. For sections to be mounted individually, I usually keep
them surrounded with a layer of 3-5mm paraffin.

If only a single specimen is embedded in a single block, trimming is easy: just shave tiny
amounts of paraffin wax off the block beginning on one side with a scalpel or a razor
blade and proceed with the other sides, until the specimen is only surrounded with a thin
layer of wax. This has to be done gently. Don’t try to cut too thick a layer of paraffin as
this might cause the block (and the specimen!) to crack.

If two or more specimens are embedded in a single block, it’s necessary to cut them out
of the block first. In my experience, the best way to do so is to gently cut a line with a
razor blade on both sides of the block about 1/3rd of the thickness of the block deep.
When that is done, the block can be broken. It will crack along the cut line. After that the
resulting blocks can be trimmed.

Trimming should be done taking the geometrical axes of the specimen into consideration:
the front side of the block should be, whenever possible, parallel with the plane of the
sections one wants to cut. If really transverse sections are to be cut, the longitudal axis of
the specimen should form a straight angle with the front side of the trimmed block in the
X as well as in the Y dimension. In other words: the specimen should be in the exact
middle of the paraffin block.

If this isn’t possible some corrections can be made by orientating the paraffin table on the
microtome, but this is only the second best thing.

The upper and lower sides of the trimmed block should be parallel. If not this will result
in curved ribbons of sections. This is especially a problem when you want to make slides
containing several serial sections.

When the specimen is hardly visible, it’s a good idea to trim the left- and/or the right side
of the block at an angle. With hardly visible specimens, this is about the only way to
recognize individual sections in the ribbon.

Left: curved ribbon due to inadequate trimming: upper and lower side of the block are not
parallel.

Middle: block trimmed okay

Right: left side of block trimmed at an angle to distinguish the sections.

Trim the rear side of the block too (the one facing the paraffin table): as paraffin is more
or less elastic, it’s necessary to trim until only a rather thin layer of wax between the rear
side of the embedded specimen and the paraffin table is left. If this layer is too thick this
can result in sections of uneven thickness.
If the specimen to be sectioned is hard you can trim the block in a pyramidal shape with
a larger basic surface. That way the block will be capable of resisting higher forces
without breaking from the paraffin table.

The resulting paraffin wax shavings from the trimming step can be used over and over
again to embed other specimens. The more the paraffin wax has been molten and
solidified, the better it gets. If needed, the paraffin wax can be filtered trough coarse filter
paper in the incubator.
 TRIMMING THE PARAFFIN BLOCK USING A SCALPEL

Before section cutting the paraffin is trimmed to expose the tissue. The cutting edge
of the knife should be parallel to the cutting surface of the block. Trimming is done by
clamping the block in the block holder of the microtone using the old knife to expose the
tissue. The block is advanced manually during this process. Another way of doing this
procedure is by increasing micron adjustment to 25 to 30 microns and rotating the hand
wheel until the tissue surface is exposed. After trimming the paraffin blocks and the knife
are cooled for 10-15 minutes in order to get good these sections. Recent microtones have
auto trim facility.

Mounting the trimmed block on a paraffin table

Usually a few paraffin tables are delivered with the microtome. When you bought a
second-hand microtome only equipped with a cassette clamp and you can’t find cassettes
you can use pieces of hardwood, aluminum, hard plastic (Stabilit, Pertinax…) sawn at the
right dimensions to fit the clamp.

My microtome came with a few paraffin tables (which I rarely use) as well as with a
“universal specimen clamp” that can hold several types/dimensions of paraffin tables.
Most of the time I use small cubes sawn from a piece of beech. These can be used over
and over again.
 PARAFFIN TABLES MADE FROM HARDWOOD, ABOUT 3CM * 2,5 CM * 1
CM

Whatever the kind of paraffin table you want to use, it has to be covered with a thin layer
of paraffin wax first. Just put a few paraffin shavings on the paraffin table and heat them
with a knife (old kitchen knife, hobby knife…) heated in the flame of a Bunsen burner or
dip the table in some molten paraffin. When necessary the table can be warmed for a few
seconds in a flame prior to applying the wax thus achieving a good bond between wax
and table.

Mounting the trimmed paraffin block on the table goes as follows: warm the knife in the
flame of a Bunsen burner. It should be moderately warm: some wax applied to it, should
melt immediately but the wax shouldn’t boil or produce smoke! Now apply the knife to
the paraffin table, put the trimmed block on it (with its upper and lower side parallel with
the sides of the block if possible as this facilitates the orientation of the block in relation
with the microtome knife) and gently pull the knife away as shown in the picture.
Stability can be optimized by melting some small paraffin shavings around the block. The
paraffin block should cool down completely.

If necessary a final trim can be done after which the paraffin block is ready to be mounted
on the microtome.
MOUNTING THE PARAFFIN BLOCK ON A PIECE OF HARDWOOD USING A
HEATED HOBBY KNIFE

Mounting the paraffin block/paraffin table and setting up the microtome

To mount the paraffin block on the microtome one just has to have a good look at the
microtome. Usually it speaks for itself just how the paraffin block/paraffin table needs to
be attached on the microtome.

It’s good practice to “turn back” the feeding screw of the microtome to its beginning after
every cutting session.
 ROTARY MICROTOME FOR LIGHT MICROSCOPY.

It is strongly advised against mounting and orientating the block while the microtome
knife is in place. For orientation purposes an empty blade holder or an aluminum strip
can be attached in the knife holder of the microtome. Once the block is properly
orientated and immediately before sectioning the knife is added.

Most microtomes have some provisions to orientate the paraffin table once it has been
attached to the microtome. In the case of my rotary microtome these are very simple:
orientation has to be done by hand only. Once you get the habit of it, it works reasonably
well.

As mentioned above: the front side of the block should be, whenever possible, parallel
with the plane of the sections one wants to cut. Viewed from aside, the surface to be cut
should be orientated parallel with the vertical movement of the specimen clamp. Viewed
from above, the surface to be cut should be orientated parallel with the knife’s cutting
edge.

If the specimen is poorly orientated within the block, adjustments should be made to
orientate it that way that the section plane is right to make the sections you want.

For real transverse sections, the longitudal axis of the specimen should form an angle of
90° with the movement of the specimen holder during sectioning. For real longitudal
sections, the longitudal axis of the specimen should be parallel with the cutting edge of
the knife. If this means that lots of sections from one side of the block (left or right, above
or below) have to be cut until (entire) sections of the specimen appear, some additional
trimming should be done.

The upper and lower sides of the block should be parallel with the microtome knife.
Setting up the microtome knife

Once the block has been orientated and mount on the microtome, the microtome knife
has to be added.

Microtome knives should be handled with the utmost respect. They are very sharp and
can produce horrible cuts. Unless section cutting, grinding or stropping is taking place,
microtome knives should always be kept in their box. In a regular household, especially
when there are little children, the knife belongs behind a locked door.

A good, sharp and well cared for good quality microtome knife should stay sharp a long
time. When the knife becomes dull it has to be resharpened on a carefully chosen honing
stone.

The knife is to be placed in the knife holder and secured with the appropriate screws.
Usually there are four screws: two to secure the knife (on the left in the picture below)
and two to secure the movable holders and the knife at the chosen angle (on the right in
the picture).

FOUR SCREWS OF THE KNIFE HOLDER

The knife should slant towards the specimen at an angle. To understand the importance of
that angle let’s first have a brief look at microtome knife geometry.

: The angle of the microtome knife’s cutting facet was always about 30°.
CROSS-SECTIONED VIEW OF MICROTOME KNIFE TYPE C SHOWING THE
CUTTING FACET’S ANGLE

MICROTOME KNIFE WITH HONING/STROPPING GUIDE AND HANDLE

ATTACHED

Determining the angle of the cutting facet of your microtome knife is very helpful when
it comes to setting up the microtome knife prior to section cutting.

An important angle to consider is the clearance angle: the angle formed between the
cutting facet of the knife and the vertical movement of the microtome.

If that angle is too obtuse, the knife won’t cut a section, but it will merely scrape a
section-alike piece from the block.

If it’s on the other hand too acute, it won’t cut a section or only a very tiny one / a
fragment as the paraffin block will be crushed between the back side of the knife’s cutting
facet and the paraffin table. After passing the knife the block expands and touches the
back side of the knife on its way up. During the next cycle a thick section will be cut as
the block has expanded again before passing the knife’s edge. This is one of the main
causes of those typical thick-thin/compressed-thick-thin/compressed ribbons.
Left: clearance angle too obtuse. The knife will scrape a section from the block rather
than cutting one. There will be very noticeable cutting artifacts. In extreme cases (as in
the sketch) the specimen will scatter

Middle: clearance angle okay, about 5°-10°, slightly more or less depending on
specimen, paraffin, temperature etc…

Right: clearance angle too acute: The paraffin block will be crushed against the rear
cutting facet of the microtome knife and the paraffin table

Given an ideal knife, the tilt of the knife should be something between 20° - 25° (thus
providing a clearance angle of 5° - 10°) and in practice this is a good starting point to cut
paraffin sections.
Most microtomes have some kind of scale on their adjustable knife holder, but it’s not
always clear what the scale is indicating. Fortunately the scale on my microtomes knife
holder is very logical: “0” means parallel with the cutting movement of the microtome.

When the knife is in place and orientated at an angle of 20°, all four screws have to be
tightened (by hand only!).

Nearly there…

The next thing to do is to move knife and specimen more closely to each other. This can
be done using the coarse feed of the microtome, the back and forth movement of the
knife holder or both. When the paraffin block is still about half or quarter of a millimeter
from the knife edge all screws of the microtome should be carefully checked and
tightened: specimen clamp, knife holder.

The section thickness dial should be set at 8-10 µm to begin with. If section cutting goes
well section thickness can be lowered to 3 - 4 µm for animal histology or slightly more (5
– 6 µm) for plant anatomy. Should it be necessary to cut thicker than 10 – 15 µm a knife
specially for that situation reserved (portion of the) should be used.

It is considered the use of a reserved portion of the knife edge or a second knife -as some
suggest- to cut thicker sections (15-25 µm) at first to reach the specimen faster an
unnecessary complication. With the embedding method I use, the specimen is usually
reached after some 40-50 sections.

When all screws are tightened you can start turning the turn wheel of the microtome at a
moderate speed of about 1 section per second

When all goes well, partial sections should occur after some rotations (given a distance
knife/paraffin block of 0,25 mm and a section thickness setting of 10 µm, sections will
occur after 25 rotations).

Unless there’s a problem, keep turning the wheel until you obtain complete sections
containing (part of the) specimen.

One of the main things that can occur is a problem with the chosen clearance angle of the
knife.

When it’s too acute, you’ll notice that there are no sections even though you see that there
should be contact between paraffin block and cutting edge. You’ll notice a strange dull
sound when the block passes the knife edge.

Have a close look at the back side cutting facet of the knife: you’ll probably see some
paraffin crushed against it. Have a look at the block too: you’ll see that part of it has -at
its sides- a white line of crushed paraffin as in the picture below on the right.
If you should cut further you’ll see that after some rotations irregular sections begin to
appear. The picture below in the middle shows that phenomenon. It’s difficult to see but
there is a sequence of compressed thin and uncompressed thin and thick sections.

The solution is obvious: slant the knife 2°-3° more towards the block and try again until
the phenomenon stops and regular sections appear. That’s the right cutting angle for that
knife, specimen, paraffin, temperature, cutting speed…

Don’t forget to move the microtome knife backwards before starting to cut again! If you
don’t, the next section you’ll cut will be a very thick one, which is disastrous for the
knife.
CLEARANCE ANGLE TOO ACUTE

Left: first partial sections.

Middle: thin-and-thick sections.

Right: crushed paraffin at the upper right corner and right side of the block
The clearance angle can be too obtuse too. In that case, sections will roll up and the knife
will produce some kind of a scraping sound. Cutting with too obtuse an angle is very bad
for the knife as well as the feeding screw of the microtome.

In extreme cases the paraffin sections show some transverse cracks. In still extremer
cases, the knife will really “bite” in the block, breaking transverse bits out of it.

The solution is obvious: slant the knife away from the block. As “biting the block” is
even worse for knife and microtome

Use a small camel hair brush to guide the sections. Never use a dissecting needle for that!
If the first section of a ribbon has a tendency to roll up, use the brush to gently hold it
with its side against the knife. The next section will stick on the previous one and a
ribbon will form when the temperature in the room is right (in my experience: about 25-
30° below the melting point of the paraffin for sections of 4-5µm and a cutting speed of
about 1 section/second). Be sure to stay well away from the cutting edge of the knife with
the brush as hairs crushed between knife edge and paraffin block can cause longitudal
striations in the sections.

Longitudal striations in the sections can also occur when the knife has small nicks in the
cutting edge. Use another part of the knife and consider regrinding it.

A third cause for longitudal striations is the knife edge being covered with paraffin or
specimen grit. In that case clean it very gently with a very soft cloth slightly moistened
with a drop of a paraffin solvent (xylene, toluene…). The pictures below show an
extreme example. These are taken during section cutting with a disposable blade but the
phenomenon is the same regardless of the nature of the knife/blade.
Too hard a tissue, due to inadequate processing. Sections are scattering. Striations in the
paraffin sections due to tissue fragments resting on the cutting edge of the blade.

If you want to mount serial sections under a common cover slip and the ribbon isn’t
straight: correct it by cutting paraffin fragments preferably from the upper side of the
block when the lower side is parallel with the knife. If the latter isn’t the case: leave well
alone when section cutting goes well or remove the block from the microtome, trim it
again and mount/orientate again on the microtome.

When these problems don’t occur or they’re corrected the section thickness dial can be
set at the right thickness.

Don’t try to cut too long ribbons: 15 or 20 cm is more than enough, unless your
microtome supports an attachable conveyor belt. Put the ribbons on a piece of cardboard
or a sheet of paper as in the picture below. Keep the ribbons in their sequential order.
Left: microtome with attached conveyor belt

Right: ribbons of sections in sequential order

When you have cut some sections, take one, put it on a slide and have a look at it under a
microscope using low and medium power. It should show no or only slight compression,
no striations and the specimen should look okay (for example: without ripped cell layers).
The pictures below show an example of a good section.
Left: Low power view in poor man’s incident light (microscope near a window) of a
paraffin section, 6 µm thick, straight from the microtome knife. Notice the small air
bubble in the paraffin surrounding the specimen. This is of no consequence as long as
these are NOT INSIDE the specimen.

Right: High power view. You’ll notice the cross sectioned stoma.

Remove the knife immediately after section cutting and clean the microtome. Paraffin
shavings can be removed with a cloth and some xylene or toluene. The microtome should
be greased according to the manufacturer’s instructions.

Section cutting using disposable microtome blades

Although I agree with the opinion that a microtomist should be able to regrind and strop
his own microtome knives, there are situations where disposable blades or razor blades
are very handy: to cut specimens of which you know they will ruin the cutting edge of
your microtome knife for example. Contrary to the situation with razor blades, the cutting
facet angle of disposable blades is indicated on their box.

In the case of disposable microtome blades, the holders they require are very expensive.
 DISPOSABLE MICROTOME BLADE HOLDER

Disposable microtome blades and their holders come in 2 versions: those for the so-called
high profile blades (dimensions 76,2 mm * 14 mm * 0,32 mm) and those for the regular
types (dimensions 80mm * 8 mm * 0,24 mm).

The disposable microtome blade holder is designed to hold the blade at an angle of about
30° - 35° towards the vertical movement of the microtome.

This means that when a blade with a cutting facet angle of 35° (that’s the standard
disposable blade, such as the Feather S/R35) is added and the knife is placed on the
microtome,in a way that the back of the blade holder is parallel with the cutting
movement of the microtome, the clearance angle will be about 0°.

One should pay special attention to the back side of the holder during section cutting: as
it protrudes towards the block it can easily scrape it.

That’s why the clearance angle for this kind of knife is usually taken somewhat larger (10
– 15°) than the one chosen when using a regular microtome knife.

Section cutting using razor blades

Razor blade holders are less expensive (about 120 Euro for the Euromex holder) than
disposable blade holders. I suppose a good mechanic can make this one without any
problem.

RAZOR BLADE HOLDER (EUROMEX)

Regarding the cutting facet angle of razor blades: it’s not that difficult to calculate it once
the thickness of the blade and the width of the cutting facet is measured. The first can be
done with a micrometer, the latter with a microscope equipped with a low power
objective and a calibrated eyepiece.

The razor blade holder I have holds the blades at an angle of about 15° towards the
vertical movement of the microtome. In that case, the blade holder needs to be tilted more
towards the paraffin block. How much more needs to be tried for every type of razor
blade to be used.

Not every razor blade is usable for section cutting. Those very thin safety razor blades are
not. Given the fact that these blades are very inexpensive, they cut at least some paraffin
embedded specimens remarkably well.

Stretching sections and attaching them onto slides

As one can imagine it’s extremely difficult to manipulate those thin sections without
damaging them. This becomes nearly impossible once the paraffin has been dissolved as
the tissue is no longer supported. That's why it’s necessary to attach the sections onto
slides.

The sections should be tightly bound to the glass surface of the slide to prevent them
from loosening during the next steps in the preparation process. Some liquids frequently
used in slide preparation are notorious for their ability to loosen sections from the slides
(as an example: ammonia water in the “bluing step” after hematoxylin staining!).

During this step the sections are stretched too, thus retrieving –more or less - their
original form and dimensions. There are several methods to achieve this.

In histological/pathological labs, sections are usually stretched in an electrical,

thermostatically controlled warm water bath, a piece of ribbon at the time. Once
stretched, the ribbon is divided into individual sections using a small brush and a
dissecting needle. The sections are subsequently picked up on slides coated with some
kind of adhesive.

Another method, which I prefer, is to stretch the sections directly onto slides using a
hotplate. This is by far the simplest way to attach series of sections on a single slide.

Pretreatment of the slides

Clean and grease free slides are absolutely necessary. New bought slides from a good
brand should be pretty clean so only minimal cleaning is required. Usually slides stored
in a mixture of equal parts ethyl alcohol and diethyl ether, rubbed with a lint free cloth
prior to use are sufficiently clean and grease free. A drop of water put on such a slide
should spread evenly.

If they are not clean (enough) it will be necessary to clean them further immediately
before use: rub them thoroughly with some detergent dissolved in hot distilled / deionised
water or in hot ethyl alcohol 10%, rinse them in hot tap water, rinse again in cold distilled
/ deionised water and store them in fresh distilled water until they are to be used. Use a
detergent for laboratory use or, if you can’t find that, the cheapest detergent you can find.
The higher grade household detergents leave a film on the glass surface containing
perfume, traces of wetting agents… Stuff that can interfere with the slide prep process.
Cheap detergents are usually a minimum formulation containing far lower amounts of
fancy ingredients.

It is strongly advised against cleaning slides with chrome-sulfuric acid. This is extremely
corrosive and poisonous –thus dangerous - stuff and from an environmental points of
view a disaster!

Stretching and attaching liquids

A thin film of an adhesive can be smeared on a slide, dried, the section applied and
stretched plain boiled and cooled down deionized water or one can stretch the sections in
a solution acting simultaneously as an adhesive. The latter method is preferred as it is less
complicated.

Haupt’s gelatin adhesive

Distilled / deionized water, about 40°C: 100 ml

Gelatin (household gelatin is satisfactory) 1 gm

Glycerin 15
ml

Thymol a
small crystal

Shelf life about a month, but it’s better to prepare a fresh solution prior to use. In that case
the thymol can be omitted.

Smear a thin film on a slide and let dry. Use boiled distilled /deionized water, cooled
down to room temperature, to stretch the sections on a slide on a hotplate or above a
flame. You should be aware of the fact that this adhesive easily picks up some regularly
used stains such as iron hematoxylin and safranin.

Use: pick up a very small amount of the solution with a clean finger tip and smear it as
thin as possible on a clean slide. Let dry. Store the slides in a dust free place.

Egg albumen adhesive

Mix one white of an egg with an equal volume of glycerin. Beat vigorously to obtain a
foamy mass. Let stand for a few hours, discard the supernatant foam. Add a crystal of
thymol.

Shelf life about a month, but it’s better to prepare a fresh solution prior to use. In that case
the thymol can be omitted.

Smear a thin film on a slide and let dry. Use boiled distilled /deionized water, cooled
down to room temperature, to stretch the sections on a hotplate or above a flame. This
adhesive easily picks up some regularly used stains such as iron hematoxylin and safranin
too, but less than Haupt’s gelatin adhesive.

Use: pick up a very small amount of the solution with a clean finger tip and smear it as
thin as possible on a clean slide. Let dry. Store the slides in a dust free place.
Unproblematic stretching / attaching liquids for home users

Distilled / deionized water, boiled and cooled down to room temperature 100 ml

(or cooled down to about 45°C when using gelatin)

Add one of the following:

Gelatin (household gelatin in sheets) 1 cm²

or

White of an egg 1
ml or

PVA (Polyvinyl alcohol) wood glue (such as Rectavit 230) 1 ml –

10 ml

This is a somewhat more water resistant (D3 according to the European norm EN 204)
white wood glue. It’s readily available in most countries I suppose, but if you can’t find it
you should try an equivalent formulation from another brand such as Bison or Titebond,
or whatever white wood glue is available in your country.

White wood glue can contain bacteria, sometimes very noticeable in the finished slides
when viewed at higher magnifications. In that case one of the other recipes should be
used or white wood glue should be tried.

Mix thoroughly. As these are easy to prepare and inexpensive I don’t think it’s
worthwhile to store them.

Use: simultaneous stretching / attaching liquid

Always use freshly boiled distilled / deionised water to prepare stretching / attaching
liquids, because the water should only contain the least possible amount of dissolved
gasses, to prevent air bubbles to occur underneath the sections.
Slide drying hotplate

As paraffin sections will only stretch when they are warmed, you’ll need some kind of
warming device. Once you have the hang of it you’ll see that it’s really no problem to
stretch sections above the flame of a Bunsen or alcohol burner, but a slide drying hotplate
is easier to use and you can stretch several slides simultaneously.

Don’t confuse this kind of hotplate with those used to boil liquids, sometimes combined
with a magnetic stirrer: a slide drying hotplate works at a much lower temperature (30 –
90°C).

If you want, it’s not that difficult to build your own hotplate. It only takes a metal cookie
tin, a bulb holder with a small electrical bulb and a dimmer.

Stretching paraffin sections only requires a moderately warm temperature of about 40 –

45 °C.

Section stretching on a slide-drying hotplate. This one is made by Jouan (France)

but you can build your own too
Stretching / attaching sections onto slides in practice

You need clean slides for this (or clean slides coated with one of the adhesives mentioned
above if you prefer that method), a pair of fine tweezers, a small camel hair brush, a
scalpel, a dissecting needle, a small pipette, stretching / attaching liquid (or distilled
/deionized water if you want to use your own adhesive - coated slides), some toilet paper
and a hotplate adjusted at a temperature of 40 – 45 °C.

Now this may sound silly, but you may want to consider wearing a surgical mask as any
uncontrolled breath can shatter your precious ribbon of sections all over the place.

Dividing the ribbon into individual sections

Perhaps you have already noticed during section cutting that the sections in the
ribbon are only loosely connected to each other, so dividing a ribbon into individual
sections isn’t that difficult. When you hold the ribbon with a fine pair of tweezers in the
middle between two sections and you put some light pressure on it with a camel hair
brush, the paraffin will usually tear between the two sections. Placing the ribbon on a
piece of paper and dividing it into sections with a scalpel or a razor blade works even
better. When the specimen is easily visible, you can even use a pair of scissors to divide
the ribbon into sections.

Make a distinction between the two sides of the section: the upper side looks somewhat
dull; the underside has a smooth and shiny appearance. It’s this side that should face the
slide during stretching / attaching it.

Stretching / attaching the sections onto slides

Take a slide and put a generous amount of the stretching / attaching liquid on it with a
pipette. There should be enough liquid to allow the section to stretch properly. Keep in
mind that paraffin sections can expand considerably during stretching (up to 25% surface
expansion). The liquid on the slide shouldn’t contain any air bubbles. Add the section
very gently, lower it down on the liquid beginning from one side to avoid air bubbles
underneath it and place the slide on the hotplate. In the course of a few seconds the
section will stretch, thus regaining its original form and size.

Once the section has stretched, hold it in place with a dissecting needle, put one side of
the slide on some toilet paper and tilt the slide to drain most of the liquid. Orientate the
section on the slide the way, wipe away the superfluous liquid and place the slide back on
the hotplate. Let dry completely.
When you use pretreated slides the method to attach sections to them is basically the
same except for the fact that freshly boiled and cooled down deionized water is used as
the stretching liquid.

When the temperature is right and there’s enough liquid under the paraffin section, it will
stretch in a few seconds only without melting
Try to avoid spilling stretching/attaching liquid on the upper surface of the paraffin
section. If this happens the section will curl up. As long as the surrounding paraffin only
is affected (as is the case here) it’s no big deal. When the slice of specimen curls up, you
could try to unfold it but in most cases the finished slide will show clearly visible folding
artifact

Stretching / attaching serial sections onto slides

It’s nearly the same as the method described above but instead of stretching individual
sections short pieces of ribbons are used.

The convention is that serial sections should be attached onto slides in the same order
Western world uses in written text: from left to right and from top to bottom.

If for whatever reason sections are missing, their place should be left open.

Paraffin section expansion should be taken into consideration as the section area of the
slide should be covered by the cover slip to be used finishing the slide. Furthermore there
should be a safety margin of at least 2 mm. (In other words: the section area of the slide
should be at least 4 mm * 4 mm smaller than the size of the coverslip.

You should keep in mind that finishing slides using large cover slips (e.g. 22 * 40mm, 22
* 50mm…) is not the easiest thing to do.

You should keep your sections/slides in a dust free slide box.

Technique
1. Place the block in the chuck of the microtome and orient it so that the top,
bottom and the face of the block are parallel to the knife edge.
2. Clamp the block firmly in position.
3. Check that the knife, knife holder and chuck are clamped firmly in position.
4. Start cutting until full sections are obtained.
5. Cut with a slow, smooth rhythm and gently pick up the ribbon of sections. The
sections are held with either a blunt forceps or a camel hairbrush or the
fingers.
6. Selected sections are floated in water bath which is maintained at temperature
just below the melting point of paraffin wax, which is used for embedding.
7. Placing them in a container having water with a few drops of alcohol will
flatten sections, a and then take them to the hot water bath.
8. Select the good section; bring an albuminized slide up to the section in an
almost vertical position. When the slide touches the section it is lifted
vertically out of water and drained. If necessary, it can be blotted in a blotting
paper.
9. Mark the slide with a diamond pencil and place the section in an oven at 60
degrees Celsius for 1 hour. Sections that need to be dried overnight should be
transferred to an oven set at 45 degree Celsius.

Tissues difficult to cut are Breast, fibrous tissue, bone, muscle, uterus and
benign tumors of these tissues.

Tissues easy to cut are Liver, Spleen, Kidney, Lymph node, and Soft tissue tumors

Ribbon sections are obtained due to the impact of paraffin wax block against the
knife edge which generates little heat just sufficient to make the sections adhere together
FROZEN SECTIONS

Sometimes in medical diagnosis it is necessary to perform a rapid analysis of a

sample. This is facilitated by performing a frozen section. The piece(s) of tissue to be
studied are snap frozen in a cold liquid or a cold environment (-20° to -70° Celsius).
Freezing makes the tissue solid enough to section with a microtome.
Frozen sections are performed with an instrument called a cryostat, a refrigerated
box containing a microtome. The temperature inside the cryostat is about -20° to -30°¡
Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then
ready for staining
Faults in paraffin section cutting and their remedies

Faults Reason Remedy

1 Sections scored vertically Knife edge is Honing .
(cut) damaged
Knife is dirty Cleaning with xylol.
2 Sections curl or roll up Knife is blunt Honing and stropping
Adjust knife tilt.
Tilt of knife is
great Boil water to increase humidity.
Static
electricity
3 Sections are alternatively Tilt of knife is Tighten adjustment screws
thick and thin(chatters) too great Adjust tilt of knife.
Tissue is very Treat the tissue with a softening
hard agent.
4 Sections crumble on Knife is blunt Sharpen the knife
cutting Wax is too soft Apply ice on the surface.
Dehydration or
cleaning is not Reprocess.
proper
5 Ribbon of section curved Block edges are Trim the edges .
not parallel to Adjust the clearance angle.
each other or to
the knife
6 Creases cannot be removed Compression of Correct the bevel by honing.
without splitting the wax the block
7 Sections lifted up from Knife edge Clean with xylol.
knife on upstroke dirty
Knife tilt less Increase tilt
Dull knife Sharpen.
8 Ribbons fail to form Paraffin too Use low melting point wax
hard Breathe on the knife.
Warm it slightly
Tilt of knife too Adjust it
great
Thick sections Cut thin sections
Dull knife Sharpen.
9 Section split vertically Damaged knife Sharpen knife
edge
Dirt in Filter the wax
embedding
media
Knife edge Clean with xylol.
dirty

Summary:
 . Paraffin blocks can be sectioned with high-carbon steel blades. Plastic blocks
(methacrylate, araldite, or epon) are sectioned with glass or diamond knives. A glass knife
can section down to about 0.1 micron. Ultrathin sections for electron microscopy (below
100 nm) are best done with a diamond knife.
Sectioning tissues is an art. The selection of knife material, blade shape, cutting
speed, knife angle and other variables must be determined through experience with the
type of tissue and the particular equipment. Sections cut under non-optimal conditions
will show tearing, ripping, 'venetian blinds', holes, folding, etc.. As sections are cut, they
are floated on a warm water bath to smooth out any wrinkles. They are then picked up on
a glass microscope slide.
The glass slides are then heated in a warm oven for about 15 minutes to help the section
adhere to the slide. This step may be bypassed to preserve characteristics such as
antigenicity. In this case, adhesive-coated slides may be substituted to pick up the
sections. Typical adhesives for this purpose include starch, albumen, resins and
combinations thereof. The adhered sections are then ready for further processing.
For Histopathological diagnosis, a good thin tissue section is very important. Well fixed
paraffin embedded block are pre requisites for getting thin sections. Instruments required
are Good microtome, Sharp microtone knife, , properly processed tissue, Block holders,
Water bath, Slide warmer, Forceps, Number 0 brush, Albuminized slides, and Experience.
Trimming is done by clamping the block in the block holder of the microtone using the
old knife to expose the tissue. The block is advanced manually during this process. Ideal
section thickness for light microscopy should be 8 microns.
Tissues difficult to cut are Breast, fibrous tissue, bone, muscle, uterus and benign
tumors of these tissues. Tissues easy to cut are Liver, Spleen, Kidney, Lymph node, and
Soft tissue tumors. Ribbon sections are obtained due to the impact of paraffin wax block
against the knife edge which generates little heat just sufficient to make the sections
adhere together.

Keywords: Microtome, section thickness, ribbon section, paraffin, micron, faults.

Questions:
1. Which is a pre requisite for getting thin sections?
2. What are the instruments used for getting a good section?
3. What is the Ideal section thickness for light microscopy?
4. Which are the tissues easy to cut?
5. Which are the tissues difficult to cut?
6. How do you obtain ribbon sections?

Answers:
1. Well fixed paraffin embedded block are pre requisites for getting thin sections.
2. Instruments required are Good microtome, Sharp microtone knife, , properly
processed tissue, Block holders, Water bath, Slide warmer, Forceps, Number 0
brush, and Albuminized slides.
3. Ideal section thickness for light microscopy should be 8 microns.
4. Tissues easy to cut are Liver, Spleen, Kidney, Lymph node, and Soft tissue
tumors.
 5. Tissues difficult to cut are Breast, fibrous tissue, bone, muscle, uterus and benign
tumors of these tissues.
6. Ribbon sections are obtained due to the impact of paraffin wax block against the
knife edge which generates little heat just sufficient to make the sections adhere
together.

IV j. HAEMATOXYLIN AND EOSIN

Histological staining involves the use of dyes to highlight specific intra- or

extracellular elements within tissue. A vast array of dyes and associated staining
protocols exist in use. Each dye is targeted toward different cellular structures. The
response to a given protocol can vary among samples. Many protocols are up to 100
years old, and were developed using partially characterized textile dyes. As a result, the
detailed mechanism underlying many popular staining techniques is unclear.

THE CHEMISTRY OF DYES:

The human eye responds to wavelengths of light between 400 and 700
nanometers (the visible spectrum). The presence of all wavelengths in this spectrum is
perceived as white light. The presence of one wavelength alone is seen as a color: Blue
for 450 nm light, Red for 600 nm light, etc. Furthermore, if one color (wavelength) is
removed from the full visible spectrum, the light is perceived as having the
'complementary color'. For example, materials which absorb at 450 nm (blue light) will
appear carmine. In general, dyes appear colored because they absorb a particular
wavelength in the visible region. The eye senses the reflected light as the complementary
color.

THE COLORS OF THE VISIBLE SPECTRUM ARE REPRESENTED ABOVE AS

THREE COMPLEMENTARY PAIRS. THE ABSORPTION OF YELLOW LIGHT
BY THE DYE EOSIN PRODUCES A COMPLEMENTARY PURPLE COLOR.
WHY DYES PRODUCE COLOR?

Absorption of light energy occurs when a compound has an electron which can be
promoted by a 'quantum permittedÓ=' mechanism to a higher energy level. The energy
difference between the ground state and the excited state determines the wavelength of
light absorbed. The energy absorbed can be re-emitted at a longer wavelength
(fluorescence), or dissipated as heat (simple absorbance). All dyes possess a
chromophore, an aryl ring system with one or more delocalized electrons. These electrons
can be promoted to excited states by visible light. The absorption wavelength of a given
ring system can be modified by the addition of non-aryl substituents (color modifiers).
For example, the successive addition of methyl groups to the red dye Pararosaniline
produces a series of dyes with progressively longer absorbance wavelengths: Methyl
violet (4 methyl groups), Crystal Violet (6 methyl groups), and Methyl Green (7 methyl
groups).

Figure 1.6.2a The molecular structures of dyes contain conjugated aromatic rings.

Figure 1.6.2b Simple absorption vs.

fluorescence.

THE CHEMISTRY OF STAINING

Staining procedures provide conditions which promote the binding of a given dye
to specific cellular organelles or extracellular features. The utility of a staining procedure
lies in its ability to bind dye only to selected structures, highlighting these structures in
contrast with the rest of the section. To accomplish this, each procedure makes use of a
subset of possible interactions between the dye and the cellular components. The major
classes of interaction (bonds) are ionic, covalent, and hydrophobic.
Ionic bonding results from the attraction between positive and negative charges.
In solution, acidic groups (carboxylic or sulfonic acids, etc.) will lose a proton and
become negatively charged (anionic). Basic groups (generally amines) will accept a
proton to become positively charged cations. The pH of the solution determines the
extent to which any chemical group is protonated or deprotonated, and a dye or biological
molecule may have many such groups on its surface. Thus, altering the pH of a staining
solution will alter the charges on the dye and the tissue molecules, and therefore alter the
staining pattern. Ionic bonds are the predominant mode of interaction between tissues and
dyes.
Covalent bonding occurs between uncharged atoms that require the gain or loss of
electrons to reach a stable configuration. In the usual scenario, the atoms involved donate
electrons to a shared orbital. The atoms then share the electrons involved, and are bonded
by the resulting orbital. Coordinate bonds are a subclass of covalent bonds in which one
of the atoms donates all the electrons (two of them) which are then shared by both of the
atoms participating in the bond. Except with mordants, covalent bonds are of little
importance in staining.
The presence of nonpolar molecules in an aqueous environment forces water
molecules to assume a highly ordered arrangement, which is entropically disfavored. A
large sphere of nonpolar molecules presents less surface area to the water than many
dispersed molecules, so nonpolar materials tend to aggregate into their own phase. This
sort of behavior, where molecules partition out of an aqueous solution, is called
hydrophobicity, and the tendency of hydrophobic molecules to self associate is called the
hydrophobic interaction. This phenomenon is utilized in staining lipids, which are
hydrophobic. Hydrophobic stains will tend to dissolve into lipid rich regions of the
section, highlighting them for analysis.
Many dyes have a poor affinity for tissue when used alone. Various compounds,
most often metal salts, have been found to enhance the staining of these dyes. These
enhancing compounds are called mordants. The mechanism of action of the mordants is
not clear, but it presumably involves coordination bonding between the metal and the
dye, and then further coordination between this complex and the tissue.

STAINING PROCEDURES

Most dyes used to visualize the membranes and organelles of the cell are water
soluble. The embedded wax must therefore be removed prior to staining. This is done by
effectively reversing the tissue processing schedule.
There are literally thousands of staining protocols and procedures in use. As an
example, one of the most common stains, the Hematoxylin-Eosin stain, is presented
below. For a detailed list of stain procedures we recommend that you visit the

Introduction:
Staining with hematoxylin is of two types – progressive and regressive. In
progressive staining, staining is continued until the desired intensity of coloring of
different tissue element are obtained. In regressive staining tissues are overstained and
excess dye is removed selectively until desired intensity is obtained.

Principles of Hematoxylin staining:

Hematoxylin is a natural dye extracted from the core or heart wood of the tree
Haematoxylon Campechianum found in Mexico.

RIPENING AGENTS:
 Natural extract obtained from the logs (hematoxylin) is not a active dye stuff. It must
be first oxidized to the active principle Hematin. Spontaneous oxidation occurs very
slowly in watery or alcoholic solution and it takes three to four months for this to be
satisfactorily accomplished. This process of oxidation is known as ripening. Ripening
can be affected instantaneously by chemical oxidants such as:
1. Mercuric oxide
2. Sodium iodate
3. Potassium permanganate
4. Hydrogen peroxide
5. Potassium per-iodate.
.
MORDANTS:

Mordants are substances which aid in attaching a stain or a dye to the tissues. They are
essential to hematoxylin staining for which the mordants used are always di- or trivalent
salts or hydroxides of metals.
The complex of stain and mordant is called as the lake. They are salts of aluminum,
iron, chromium, copper, molybdenum, and vanadium.
1. Potash alum
2. Iron alum
3. Ammonium alum
Aluminum salts give a blue lake, whereas ferric salts form intense blue black.

DIFFERENTIATION:

Staining with hematoxylin and many other dyes may be progressive or regressive. In the
regressive method the excessive stain is removed selectively until the right intensity is
obtained. This is called as differentiation.
Differentiators for mordant dyes is classified as
1. Acid differentiators
2. Oxidizing differentiators
3. Mordant differentiators.

Acid differentiator combines with metal and breaks the union of metal with tissue or cell
component. Acid chosen should be one which forms a soluble salt with the metal so that
the latter is dissolved out. E.g.: Hydrochloric acid, acetic acid.

Oxidizing differentiator oxidize the dye to colorless substance. E.g.: potassium

ferricyanide, potassium permanganate, and picric acid.

Mordant differentiator acts by a phenomenon of mass action. Excess mordant will help in
the removal of excess stain.

BLUEING:
Process in which the free acid is neutralized and insoluble blue aluminium Hematin
Tissue Lake is formed. Blueing solutions used are
1. Lithium carbonate
2. Bicarbonate
3. Potassium or sodium acetate
4. Scott’s tap water substitute
5. Tap water if sufficiently alkaline
Few crystals of thymol or 5-10 ml of formaldehyde is added to prevent the growth of
moulds.

TYPES OF HEMATOXYLIN:
1. Alum hematoxylin
2. Iron hematoxylin
3. Tungsten hematoxylin
4. Lead hematoxylin
5. Molybdenum hematoxylin
6. Hematoxylin without mordant.

Alum hematoxylin
Those are the most routinely used hematoxylin to produce good nuclear staining.
Mordant is aluminium in the form of ammonium alum or potassium alum. Nuclear stain
red which are converted to blue black by washing in a weak alkali or tap water. Scott’s
tap water is frequently used for bluing.
Most widely used alum hematoxylins are Harris hematoxylin, Ehrlich hematoxylin,
Mayer’s hematoxylin and Cole’s hematoxylin.

Iron hematoxylin:
Iron salts used are ferric chloride and ferric ammonium sulphate (iron alum). Iron salts
serve as both oxidizing agents and mordants. The ferric salts oxidize the hematoxylin
chemically and should not be kept for too long. Ideally, it should be prepared
immediately before use. Iron hematoxylins demonstrate a wider range of tissue structures
than the alum but are more time consuming.
Most widely used iron hematoxylins are the Weigert’s, Verhoeff, Heidenhain, Loyez
hematoxylin.

Preparation of Stains

Harris’s alum Hematoxylin

1. In 1000 ml of distilled water in a large (3-4 liter) Erlenmeyer flask dissolve 100g
of ammonium or potassium alum by heating and shaking.
2. Bring to 60˚C, add a solution of 5g of hematoxylin in 50 ml of absolute ethyl
alcohol and bring rapidly to the boil
3. When it begins to boil, remove from flame and add 2.5g of mercuric oxide. Mix
by swirling gently.
 4. The solution immediately becomes purple and most workers suggest that solution
must be immediately cooled by plunging the container into a sink filled with cold
water.
5. When cooled 20 to 40 ml of glacial acetic acid is added and stain filtered before
using.
6. Add 50 ml of ethyl alcohol to the final solution; it helps to prevent the growth of
moulds.

Features of Harris hematoxylin:

1. Good regressive stain
2. easily made
3. Immediately ready for use.
4. Stable for 6 months. Acid gives stability to the stain and improves nuclear stain.
5. Staining time for Harris hematoxylin varies between 4-30 min depending on the
batch of the stain, its age, nature of tissue and depth of staining required.
6. Best results are obtained if the formula is made every 2-3 months.

EOSINS:

Eosins are acid xanthene or pthalein dyes. Eosin Y, Eosin b, phloxine and erythrosine
are the common members of this group of dyes. Eosin is derived from fluorescein and is
available in two shades- yellow and blue. It is most commonly employed as a contrast
stain because it gives a useful differential contrast to nuclear stains. Eosin derives its
name from its dawn like color and Y stands for yellowish which is its predominant shade
of red.
Eosin Y is the most frequently used and is readily soluble.(44 ℅ w/v in water and 2
℅ w/v in ethanol). add a crystal of thymol or 1% formalin to prevent growth of molds.
The aqueous stain is usually used at 1 ℅ for 30 seconds to 5 minutes, depending on the
type of fixative, tissue and intensity of color desired.
Alocholic solution (1% w/v) is made by dissolving 1g in 20 ml distilled water and
adding 80 ml of absolute or 95% ethanol.
Eosin is most commonly used counterstain in hematoxylin staining methods.
However, many substitutes are available, e.g., phloxine, Biebrich scarlet, erythrosine, and
orange G. These substitutes are prepared in similar concentrations and modes as eosin.

Routine H&E Procedure;-

1. Dewax and put the slide in xylene to remove wax (To deparaffinize) 6-8 min.
Excess solution should always be drained.
2. Wash off the xylene with absolute alcohol 3-4 min.
3. Rinse in running tap water for 3-4 minute
4. Stain with Harris Hematoxylin for 4-8 minutes.
5. Wash thoroughly with tap water (15 - 30 seconds)
6. Differentiate in 1% acid alcohol for 10 -20 seconds (HCL 1ml +80% ethyl alcohol
99ml)
 7. Wash with tap water.
8. Blueing (Process when a section is transferred from an acid solution to an alkaline
solution) –Section is placed in cold tap water for 5-10 minutes.
9. Counterstain with 1% aqueous eosin for 1-3 minutes.
10. Wash in tap water.
11. Wash with 95% alcohol for a few seconds.
12. Clear by washing with xylene.
14. Mount in Canada Balsam or DPX.

Note the use of tap water in the washing steps - tap water provides the alkanlinity
necessary for the "bluing" process.

Results;-
Cell nuclei - Blue.
Cytoplasm, proteins in edema – pale pink.
Muscle fibres, erythrocytes and eosinophil granules – bright Red.
Collagen fibres – Pink
Bone and calcium – browner and less intense.

Methods of saving hematoxylin:

1. Celestine blue B can be used as an alternative to hematoxylin.
2. Using Mayer’s hematoxylin instead of Harris yields a saving of 4/5 in the amount
of dye used.
3. A 30 second wash in 0.5% aqueous citric acid before staining in hematoxylin will
prolong the staining life of the hematoxylin solution
4. Overoxidation of the hematoxylin solution may be retarded by keeping a lid on
the stain container when the stain is not in use. Oxidation by light is avoided by
placing a cloth over the staining container when it is not being used.
5. Substitute special stains which employ dyes other than hematoxylin, e.g. Luxol
fast blue instead of Loyez for myelin.
6. After a solution of Harris hematoxylin has reached the end of its staining
usefulness, allow to evaporate tot about half its volume. Then add 10 ml saturated
aqueous ammonium aluminium for each 100 ml of hematoxylin solution. Filter
after 24 hours and employ in staining of tissues doubling the usual time.

NOTE: From the outset, each and every step of staining must be controlled by
microscopic examination.

SUMMARY:
Hematoxylin is a natural compound extracted from a species of tree found in
Mexico and the West Indies. The extracted compound is then oxidized to produce
hematein, which is the active staining component of the hematoxylin stain. Hematoxylin
stains must therefore be 'ripened' by oxidation before they can be used. This process of
oxidation is known as ripening. Ripening can be affected instantaneously by chemical
oxidants such as: Mercuric oxide, Sodium iodate, Potassium permanganate, Hydrogen
peroxide, and Potassium per-iodate.
Types of Hematoxylin are Alum hematoxylin, Iron hematoxylin, Tungsten
hematoxylin, Lead hematoxylin, Molybdenum hematoxylin and Hematoxylin without
mordant.
Staining with hematoxylin is of two types – progressive and regressive. In
progressive staining, staining is continued until the desired intensity of coloring of
different tissue element are obtained. In regressive staining tissues are overstained and
excess dye is removed selectively until desired intensity is obtained.
. Hematoxylin staining requires the use of a mordant (most commonly aluminum
salts) and stains the nuclear components of cells a dark blue. Mordants are substances
which aid in attaching a stain or a dye to the tissues. The complex of stain and mordant is
called as the lake. They are Potash alum, Iron alum, and Ammonium alum
Staining with hematoxylin and many other dyes may be progressive or regressive.
In the regressive method the excessive stain is removed selectively until the right
intensity is obtained. This is called as differentiation. Differentiators for mordant dyes
are classified as Acid differentiators, Oxidizing differentiators, and Mordant
differentiators.
Blueing is the process in which the free acid is neutralized and insoluble blue
aluminium Hematin Tissue Lake is formed. Blueing solutions used are Lithium
carbonate, Bicarbonate, Potassium or sodium acetate, Scott’s tap water substitute, and
Tap water if sufficiently alkaline.
Alum hematoxylin is the most routinely used hematoxylin to produce good
nuclear staining. Most widely used alum hematoxylins are Harris hematoxylin, Ehrlich
hematoxylin, Mayer’s hematoxylin and Cole’s hematoxylin. Most widely used iron
hematoxylins are the Weigert’s, Verhoeff, Heidenhain, Loyez hematoxylin.
Hematoxylin is used in combination with eosin because eosin stains the
cytoplasmic organelles varying shades of pink, red or orange. The combination of the two
stains provides a broad range of morphological information about the section.
Eosins are acid xanthene or pthalein dyes. Eosin Y, Eosin b, phloxine and
erythrosine are the common members of this group of dyes. Eosin is derived from
fluorescein and is available in two shades- yellow and blue. Eosin is most commonly
used counterstain in hematoxylin staining methods. However, many substitutes are
available, e.g., phloxine, Biebrich scarlet, erythrosine, and orange G.
Results of H&E procedure are --- Cell nuclei - Blue, Cytoplasm, proteins in
edema – pale pink, Muscle fibers, erythrocytes and eosinophil granules – bright Red,
Collagen fibers – Pink, and Bone and calcium – browner and less intense.

KEYWORDS: Hematoxylin, alum, Harris, Eosin, Mordant, Iron Hematoxylin.

QUESTIONS:
1. What is Hematin?
2. What are the types of Hematoxylin?
3. What are the types of Eosin?
4. What are the substitutes for Eosin?
5. What are the types of iron hematoxylin?
 6. How do you save hematoxylin?
7. What are mordants?

ANSWERS:
1. Hematoxylin on oxidation becomes haematin which is the essential staining
element.
2. Types Of Hematoxylin are Alum hematoxylin, Iron hematoxylin, Tungsten
hematoxylin, Lead hematoxylin, Molybdenum hematoxylin and Hematoxylin
without mordant.
3. Eosin Y, Eosin b, phloxine and erythrosine.
4. phloxine, Biebrich scarlet, erythrosine, and orange G.
5. Weigert’s, Verhoeff, Heidenhain, Loyez hematoxylin.
6. Methods of saving hematoxylin: Celestine blue B can be used as an alternative to
hematoxylin. Using Mayer’s hematoxylin instead of Harris yields a saving of 4/5
in the amount of dye used. A 30 second wash in 0.5% aqueous citric acid before
staining in hematoxylin will prolong the staining life of the hematoxylin solution.
Overoxidation of the hematoxylin solution may be retarded by keeping a lid on
the stain container when the stain is not in use. Oxidation by light is avoided by
placing a cloth over the staining container when it is not being used. Substitute
special stains which employ dyes other than hematoxylin, e.g. Luxol fast blue
instead of Loyez for myelin. After a solution of Harris hematoxylin has reached
the end of its staining usefulness, allow to evaporate tot about half its volume.
Then add 10 ml saturated aqueous ammonium aluminium for each 100 ml of
hematoxylin solution. Filter after 24 hours and employ in staining of tissues
doubling the usual time.
7. Mordants are substances which aid in attaching a stain or a dye to the tissues.
They are essential to hematoxylin staining for which the mordants used are
always di- or trivalent salts or hydroxides of metals.

IV k. Mounting Medias-

INTRODUCTION:
Sections are mounted under cover slips to maintain a high refractive index
necessary for a microscopy and to protect sections during storage. To preserve and
support a stained section for light microscopy, it is mounted on a clear glass slide, and
covered with a thin glass coverslip. The slide and coverslip must be free of optical
distortions, to avoid viewing artifacts. A mounting medium is used to adhere the coverslip
to the slide.
Aqueous based mounting media are available, which allow the mounting of
tissues directly from the staining procedure. However, the water solubility of some stains
allows them to bleed and/or fade in such mountants, necessitating the use of resinous
mounting media. To use a nonaqueous mountant, the section must first be dehydrated
(again!) and cleared. Any water carried over to the mounting stage will show up as
bubbles or vacuole-like structures, as the water droplets aggregate and distort the tissue.
It is important to note also that the clearing agent used must be compatible with
the mounting medium, or the sections must be thoroughly dried prior to mounting.
 Mounting Medias – two types
1. Aqueous media
2. resinous media

Aqueous media – routinely used, have low refractive index hence resinous medias are
used routinely. They are semi-permanent mountants.
• Gelatin media
• Gum Arabic media
• Glycerine jelly media
• Apathy’s media
• Farrant’s media
• Fructose syrup
• Water
• Mineral oil

Sealing temporary and semi-permanent mounts can be done using paraffin wax, Kronig
mixture, Nail varnish etc.

Mounting thin plastic sections is done using Harleco synthetic resin (HSR).

Resinous media – these media are composed of resin, natural or synthetic

• Canada balsam
• Dammar balsam
• Terpene resin
• Synthetic resins – Distrene Plasticizer Xylene (DPX)

The most common synthetic resins are the polyesterenes, such as Kirkpatrick and
Lendrums, DPX.
Composition –
Distrene 80 10 grams
Dibutylphthalate 5 ml
Xylol 35 ml
Advantages –
• Excess mountant is removed easily by cutting and stripping it around the
edge of the cover slip
• Excellent mounting media with refractive index 1.52.

Other mounting medias are –

• Euparal
• Ringing media
 MOUNTING MEDIA
Qualities of a good mounting media:
1. It must have a refractive index close to that of glass (1.518).
2. It must be freely miscible with xylene and toluene.
3. It must be non-reactive.
4. It will not change color or pH.
5. It will set hard without granularity or cracking.
6. It will not leak out any stain.
7. It should not cause loss of staining over long periods.

TECHNIQUE FOR MOUNTING SECTIONS:

1. Following clearing in xylene, clean a coverslip of the appropriate size and place it
on a white blotting paper, preferably on a sheet of Whatman no. 1 filter paper.
2. With a blunt forceps, pick up the slide carrying the section from the xylene bath,
clean the ends first so that the diamond inscribed number is visible and the front
of the slide is identified.
3. Then wipe the back of the slide with a clean dry and soft dust free cloth.
4. With a small glassrod, place the necessary amount of mounting media on the
section.
5. Quickly invert the slide and lower it onto the coverslip, applying gentle pressure.
As soon as the medium comes in contact with the coverslip it spreads evenly to
the edge of the coverslip and covers the whole area of the section which should
still be moist with xylene.
6. Finally the slide is labeled indicating the tissue type or stain used and specimen
number.
7. Following this sections are ready to be examined. Care should be taken not to
move the coverslip. Usually , the slides should be placed on hot plates ( 500 c) or
 in the wax oven for up to 2 hours or at 37 0 overnight. This is to harden the
mounting media.

Fig . METHOD OF MOUNTING A COVERSLIP

Fig . Mounting media can be taken either on coverslip or slide.

Coverslipping - Important considerations:

1. Choose a proper size coverslip for specimen.
2. Roll coverslip onto slide to expel trapped air.
3. Clean excess mounting media from the edges of coverslip with gauze moistened
with appropriate solvent. Avoid smearing the viewing area.

PRECAUTIONS DURING MOUNTING:

Air bubbles are formed due to less of media used. When there is more than one air
bubble in the mountant it is quicker to put the slide back into xylene and remount the
section. An odd air bubble when present, it may be expressed by gentle pressure on the
coverslip with a dissecting needle.
 DPX should be used more liberally than any other mountants as it causes
retraction on drying.

Some mounting medias:

1. Hydromount is a water-based synthetic resin, containing glycerin, suitable for
mounting specimens which have been processed in water. Hydromount can be used
for frozen sections as well as amyloid and Immuno fluorescent staining procedures.
2. Plastic UV Mount is designed to match the refractive index of JB-4 embedded
sections thus improving the final cover glass mount. Hardens permanently within
minutes when exposed to long wave UV light.
3. Entellan is a Colorless - rapid mounting media for microscopy. Very good mounting
media for long life preparation. There is no bubble formation at high ambient
temperatures. The cure time is 20 minutes at room temperature.
4. Eukitt is a quick-hardening mounting medium possessing physical, chemical and
optical properties which make it ideal for slide preparations. It is neutral, colorless
and spread quickly and evenly. Refractive index same as glass. In addition is useful
for sealing cover slip over wet preparations.
5. SHUR/Mount is a unique water based mounting medium for use in all Immuno
procedures. Designed for cover slipping wet slides. Available in 20 ml bottle.
6. DPX Mountant for Microscopy is a colorless, synthetic resin
mounting media. Replaces Xylene-Balsam mountant. It
preserves the stain and dries quickly.

7. Histomount is a non-hazardous synthetic mountant. It has

neutral pH, is UV stabilized and is effective with most
clearing agents when used as a liquid cover slip seal.
8. Mount Quick -Water Base is a mounting media for cover
glass. It was developed for mounting stained sections for
lipoid and immune staining. It comes in a convenient 30 ml
bottle.
9. Mount Quick – Solvent base

is
is a solvent base mounting media comes in a tightly capped tube. It is soluble in
xylene. Mount Quick dries quickly (10 minutes) and not forming bubbles.
10. Quick-Stick® Mounting Medium - This mounting medium is available in a
convenient stick form that can be applied to a slide on a hot plate. Once the
specimen and cover glass are positioned and the slide is cooled, a permanent
preparation is formed. The process can be reversed by reheating the slide.
11. Biomount™ - Tissue Section Mounting MediumBiomount™ mounting media is
specially formulated to reduce fading of Immuno gold/silver signals in sections on
glass slides. It is suitable for wax or resin embedded sections of tissue.
12. Citifluor Antifadent Mountant Media

The photofading of fluorescein-labelled materials can be retarded by the

use of the Citifluor mountants.

Three types are available.

AF1 Is a Glycerol-phosphate buffered solution

containing additives for use with labeled tissue.

AF2 Is a Glycerol solution containing additives for use with labeled tissue
sections

AF3 Is a Phosphate-buffered saline solution containing additives for

examination of whole cells.
Available in 25 ml bottle.

SUMMARY:

Sections are mounted under cover slips to maintain a high refractive index
necessary for a microscopy and to protect sections during storage. Mounting Medias are
of two types-Aqueous media and resinous media.
Aqueous -Gelatin media, Gum Arabic media, Glycerine jelly media, Apathy’s media,
Farrant’s media, and Fructose syrup. Resinous media – Canada balsam, Dammar balsam,
Terpene resin, Synthetic resins – Distrene Plasticizer Xylene (DPX). Other mounting
medias are –Euparal, Ringing media. Media used routinely is DPX. Qualities of a good
mounting media are ,it must have a refractive index close to that of glass (1.518), freely
miscible with xylene and toluene, non-reactive, will not change color or pH, set hard
without granularity or cracking, not leak out any stain, should not cause loss of staining
over long periods.

KEYWORDS: mountants, aqueous, resinous, DPX, media

QUESTIONS:
1. What is the purpose of mounting?
2. What are the types of mountants?
3. What are the advantages of DPX?
4. What are the properties of an ideal mounting media?

ANSWERS:

1. Sections are mounted under cover slips to maintain a high refractive index
necessary for a microscopy and to protect sections during storage.
2. Mounting Medias – two types aqueous media and resinous media
3. Excess mountant is removed easily by cutting and stripping it around the edge of
the cover slip. Excellent mounting media with refractive index 1.52.
4. Qualities of a good mounting media are ,it must have a refractive index close to
that of glass (1.518), freely miscible with xylene and toluene, non-reactive, will
not change color or pH, set hard without granularity or cracking, not leak out any
stain, should not cause loss of staining over long periods.