TISSUE PROCESSING

MLS 301
“Tissue processing,” describes the steps required to take an animal or human tissue from fixation to the
state where it is completely infiltrated with a suitable embedding medium and subsequent section
cutting on the microtome.
Tissue processing can be performed manually (hand processing), but where multiple specimens must be
dealt with, it is more convenient and much more efficient to use an automated tissue processing
machine (a “tissue processor”).
These devices have been available since the 1940’s and have slowly evolved to be safer in use, handle
larger specimen numbers, process more quickly, and to produce better quality outcomes.

There are two main types of processors:

1. The tissue-transfer (or “dip and dunk”) machines where specimens are transferred from container to
container to be processed.
2. The fluid-transfer (or “enclosed”) types where specimens are held in a single process chamber or
retort and fluids are pumped in and out as required.
Most modern fluid-transfer processors employ raised temperatures, effective fluid circulation and
incorporate vacuum/pressure cycles to enhance processing and reduce processing times.
Completion Of Fixation
Fixation is the most important step in the processing of tissue sample. If fixation
is not complete prior to processing, stations should be designated on the
processor for this purpose. If tissue is inadequately fixed, the subsequent
dehydration solution may complete the process, possibly altering the staining
characteristics of the tissue.

Post-fixation Treatment
Special fixation techniques may require additional steps before processing is
initiated. For instance, picric acid fixatives form water-soluble picrate making it
necessary to place the tissue cassettes directly into 70% alcohol for processing.
Alcoholic fixatives, such as Carnoy’s fluid, should be placed directly into 100%
alcohol. To help in the visualization of small fragments of tissue during
embedding a few drops of 1% eosin can be added to the specimen container
30mins prior to processing. The pink coloration of the tissue remains during
processing. However, washes out during subsequent staining.
Principles Of Tissue Processing
This is based on the removal of all extractable water from the tissue and
replacing it with a support medium that provides sufficient rigidity to enable
sectioning of tissue without damage or distortion.
Factors Affecting The Rate Of Processing
Agitation; this increases the flow of fresh solutions around the tissue,
efficient agitation can reduce the overall processing time by 30%
Heat; heat increases the rate of penetration and fluid exchange, temperature
limited to 45℃ can be used effectively higher temperature may be
deleterious to subsequent staining
Viscosity; viscosity is a property of resistance to the flow of fluid the lower
the viscosity of the fluid in use the higher the rate of penetration, most of the
solution used in processing, dehydrates and clearants have similar viscosity
with expection of cedar wood oil. Embedding mediums have varying
viscosities. Paraffin has lower viscosity in fluid state, this enhancing the
rapidity of the impregnation.
Vacuum; using reduced pressure to increase the rate of infiltration decreases
the time necessary to complete each step in the processing of tissue sample,
vacuum will remove reagents from the tissue only if they are more volatile
than the reagent to replace with.
Stages Of Tissue Processing
Dehydration
This is the first stage of processing, removal of unbounded water and
aqueous fixatives from tissue components. Dehydration should be
accomplished gradually, if the concentration gradient between the fluid and
the tissue is excessive, diffusion currents cross the cell membrane during
fluid exchange increasing the possibility of cell distortion. Excessive
dehydration may cause the tissue to become hard, brittle and shrunken. On
the other hand incomplete dehydration will prohibit the penetration of the
clearing agent into the tissue, leaving the specimen and non-receptive to
infiltration. There are numerous dehydrating agents; acetone, methanol,
isopropyl, glycol and denatured alcohol. If the dehydrant of choice is
ethanol the tissue is first immersed in 70% in water, then 95% and finally
100%.

Clearing
The clearing agents are miscible with both dehyrants and infiltration
solution. Clearing agents renders the tissues translucent because the
refractive index of clearing agents is approximately equal to that of tissue
proteins.
•Why is clearing required during processing

•Clearing is required to remove alcohol from tissues and is replaced by fluid

which is miscible with wax with which tissue must be impregnated
•Clearing agent is necessary as the dehydrating agents are not miscible with
paraffin
•Clearing agent is miscible with both dehydrating agent as well as paraffin wax

•Criteria for choosing a suitable clearing agent

• Good clearing agent should rapidly remove dehydrating solution
• Minimal tissue damage
• Should not dissolve out aniline dyes
• Cost effective
Different type of clearing agents
 Benzene
 Xylene
 Toluene
 Choloroform
 Petroleum ether
 Oil of wintergreen (Methyl salicylate)
 Cedar wood oil
 Carbon tetrachloride
 Clove oil
 Dioxane
 Aniline oil
Xylene
Advantages of xylene are
It is cheap and rapid in action
Can be used in most of tissues
Can be used for both paraffin and celloidin embedding

What are the disadvantages of xylene

It makes tissues excessively hard and brittle, if the tissue is left for more
than 3 hours in xylene
It is not suitable for brain and lymph nodes
Should be handled with care as it causes dermatitis

How do we know whether dehydration is complete or not

Pour the xylene on the tissue section. If the dehydration is not complete
then the xylene becomes milky. Then the tissue should be placed again in
absolute alcohol
Toluene
This is also used for clearing in tissues for electron microscopy
The advantage and disadvantage of toluene as clearing agent
Toluene causes less hardening than xylene but the clearing is much slower than
xylene
Chloroform
This is an excellent clearing agent for nervous tissue, lymph nodes and embryos
as it causes little shrinkage
It does not harden tissue excessively
It is best clearing agent for large blocks
Not inflammable
The disadvantages of choloroform
It is expensive and evaporates rapidly from wax both
Benzene
Benzene is toxic and highly flammable
Cedar wood oil
Wax impregnation after cedar wood oil is slow and 3 changes are essential
Oil of Wintergreen (Methyl salicylate)
This clearing agent is used for making embryos, plants and insects transparent
so that their internal structures can be seen
 Procedure of clearing
After dehydration of tissue, it is immersed in 2 or 3
changes of xylene
At the end of clearing tissue should look transparent.
A typical clearing sequence for specimens not more than
4mm thick would be:
xylene 20 min
xylene 20 min
Xylene 45 min
INFILTRATION/IMPREGNATION and EMBEDDING
Infiltration is the saturation of tissue cavities and cells by a supporting substance
Which is generally, but not always, the medium in which they are finally
embedded. It is the process of replacing clearing agent by embedding media; tissues are
infiltrated by immersion in a substance such as a wax, which is fluid when hot and solid
when cold.
The tissues, after fixation and dehydration process, are not sufficiently hard to cut into
thin sections without a suitable support. Thus, tissues are first impregnated and then
embedded in a suitable embedding medium to make a block which makes the tissue quite
hard and provides a suitable support so that it can easily be cut into thin sections of few
microns. The impregnation and embedding of tissues are done by using the same medium
usually but sometimes two different mediums are used for these processes (Double
embedding). The block may be made harden by cooling it at room temperature or in the
refrigerator. There are various types of embedding medium used in the histopathology
laboratory as per the properties of tissue and the tests to be done.
PROPERTIES OF AN IDEAL EMBEDDING MEDIUM
The embedding medium is considered as ideal if it bears the following qualities:
The melting point of the embedding medium in the pure state should be below 65°C to
avoid damage to the tissues by heating during impregnation.
The embedding medium must be solidifying at room temperature.
The embedding medium must penetrate the tissues replacing the fluid in which it is
saturated, usually a clearing agent.
VARIOUS TYPES OF EMBEDDING MEDIUM
1.) Paraffin wax – Paraffin is solid at room temperature. The melting point of paraffin ranges
from 40-60°C. For tropical countries hard wax having a melting point of 58-60°C is suitable.
This is the most commonly used tissue embedding medium.
2.) Paraplast – This is a mixture of purified paraffin and plastic. It is easy to make the serial
sections of 4 microns with this embedding medium due to its elasticity.
3.) Gelatin – Water soluble and so dehydration and Clearing is not necessary. It is used for
delicate tissues and for the frozen sections. It has low melting point.
4.) Ester wax – It is harder than paraffin but has a low melting point of 46-48°C. It is soluble
in alcohol so there is no need for the clearing step when using this tissue embedding
medium.
5.) Tissue mate – It is the mixture of paraffin and rubber and has similar properties as that of
Paraplast. The serial sections can easily be obtained using this embedding medium.
6.) Water-soluble waxes – These are Polyethylene glycol, having a melting point of 38-42°C.
They cause less shrinkage than paraffin and no dehydration and clearing is needed. They do
not make the tissues brittle & give better support than paraffin. They are used for
demonstration of lipid and enzymes.
7.) Celloidin – This is purified nitrocellulose and is used for the hard and fragile specimens.
Large specimens can easily be sectioned using this embedding medium. It takes 2-3 weeks to
impregnate the tissues with Celloidin.
THE PARAFFIN WAX
Paraffin is a mixture of hydrocarbon produced in the cracking of paraffin (a
mineral oil). The melting point of high-quality paraffin ranges 40-60°C.
Paraffin wax is the most popular embedding medium because of its
properties and a large number of blocks can be prepared in a less amount of
time. It also aids in the sectioning process and does not interfere with the
staining process.
A series of sections cut from the wax block adhere to each other to form a
ribbon, these sections are easier to handle than a single section. Paraffin
Wax is on advantage here over other tissue embedding media like
nitrocellulose.
As a precaution, the wax should be free from dust and impurities and the
wax must be rapidly cooled to reduce the size of the crystals of the wax.
IMPREGNATION OF THE TISSUES
After Clearing, the tissues are infiltrated with a supporting medium which
also replaces the clearing agent. The supporting medium will make the tissues
firm, facilitates easy sectioning and keep the various components of the
tissues in proper relation.
In this step, two processes take place simultaneously i.e. the Impregnation of
the paraffin wax into the tissues and the Infiltration of Clearing agent out of
the tissue into the surrounding. In this ways, the clearing agent is replaced by
embedding medium.
This process is also called as the internal embedding of the tissues as the
embedding medium penetrates the tissues and provides the support from
inside of the tissue.
The Impregnation of tissues with molten paraffin is done in an embedding
bath with a thermostat and a vacuum pump attached. The vacuum produced
by vacuum pump increases the rate of wax impregnation and the paraffin wax
penetrates the tissues more effectively. Paraffin of high melting point
becomes hard and the paraffin of low melting point remains soft after
cooling.
A typical infiltration sequence for specimens not more than 4mm thick would
be:
wax 30 min
wax 30 min
THE TISSUE EMBEDDING OR BLOCK PREPARATION
In this, the wax impregnated tissues are placed in a mould in which molten
paraffin wax is poured, which when is solidified provides a hard, solid support to
the tissue so that it can easily be cut into the slices.
When performing the embedding of tissues, one should keep certain factors in
mind
the most important one is the choice of paraffin of the right degree of hardness,
which depends on the following factors –
i.) Nature of the object/tissue (i.e. Hard or Soft)
ii.) Temperatures at which the tissues are sectioned.
iii.) Thickness of the section
MOULDS
A variety of moulds are used for embedding the tissues but the most popular one
is Leuckhard L-shape embedding moulds, popularly known as the embedding L’s.
Nowadays, the inexpensive plastic moulds are replacing the other types of
embedding containers. These plastic moulds support the blocks while it is being
sectioned and are designed to fit according to the microtomes, eliminating the
step of mounting the specimen on a block holder.
MICROTOMY

 Microtomy is a technique in which tissue are sectioned and attached to a

surface for further microscopic examination. The basic instrument used in
microtomy is the Microtome
 The first microtome suitable for sectioning animal tissue was constructed in
1848 the popular Cambridge rocker, Minot(the rotary microtome (1886) and
sledge microtomes (1910).
 The microtome use steel, glass or diamond blades depending on the specimen
being slice and the desired thickness of section being cut, steel blade is
mostly used to section tissue for light microscope while glass knifes are used
mainly for electron microscope and also light microscope.
 The diamond blades are used for hard materials such as bone teeth and tough
plants. Various types of microtomes are available. Most commonly used
microtome for routine histopathology is rotary microtome.
Types of microtome

 1. Rotary microtome
 2. Base sledge microtome
 3. Rotary rocky microtome
 4. Sliding microtome
 5. Ultra microtome
 6. cryostat
Rotary Microtome – also referred to as Minot after the inventor, it is most commonly used
microtome.
This device operates with a staged rotary action such that the actual cutting is part of the
rotary motion.
In a rotary microtome, the knife is stationary at a horizontal position and a rotary action
of the hand wheel actuate the cutting movement.
It has an advantage over the rocking type due to its weight, which makes it stable with
the possibility to cut hard tissues without vibration; this also enhances easy Serial sections
or ribbons of sections.
The block holder or block (depends upon the type of cassette) is mounted on the steel
carriage that moves up and down and is advanced by a micrometer screw.
The rotary microtome can be manual (completely manipulated by the operator)
semi-automated (one motor to advance either the fine or coarse hand wheel, this has in-
built motor drive with foot, and hand control) or fully automated (two motor that drives
both the fine and coarse advance hand wheel).
With suitable accessories, the machine can cut thin sections of paraffin wax blocks and
0.5 to 2.0 micrometer thin resin sections.
Principle of a rotary microtome

 The basic mechanism requires the rotation of fine advance hand-wheel

through 360◦
moving the specimen vertically past the cutting surface and returning it
to start position.
Advantages

 The machine is heavy, so it is stable and does not vibrate during cutting.
 Serial sections can be obtained.
 Cutting angle and knife angle can be adjusted.
 It may also be used for cutting celloidin embedded sections with the help of
special holder to set the knife.
The Base Sledge Microtome
 In this device samples are placed stationary into a fixed holder (shuttle) and the knife slides
across the top of the specimen during processing.it is used primarily for large blocks, hard tissue
or whole mount, it is very useful in neuropathology and ophthalmic pathology. 3 micron are
difficult to produce. The typical cut thickness achievable on a sledge microtome is between 10
and 60 micron.
The rotary rocking microtome
Common in early cryostats, the retracting action moves the tissue block away from knife on
the upstroke, producing a flat face to the block

The sliding Microtome

The knife or blade is stationary and the specimen slides under it during sectioning. This
microtome was mades purposely for celloidin-emdedded tissue block

Cryostat microtome
For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a
liquid nitrogen chamber, in a so-called cryomicrotome setup. The reduced temperature
allows for the hardness of the sample to be increased, such as by undergoing a glass
transition, which allows for the preparation of semi-thin samples. However, the sample
temperature and the knife temperature must be controlled in order to optimize the
resultant sample thickness
Ultra-microtome
This type of microtome is mainly for sectioning of samples viewed under electron
microscope, it produces ultrathin sections (20-150nm thick) in a fast and clean manner. The
key advantage of ultra-microtome is the size and homogeneity of electron transparent area
within the section and the speed at which the section are produced.
The sliding Microtome
The rotary rocking microtome
Cryostat Microtome
Ultra-microtome
 MICROTOME KNIFE
There are many shapes, sizes, and materials for microtome knives, the knives developed to fit
specific types of microtome and equally cope with different degrees of hardness of tissues and the
embedding medium used. Microtome knives are made of good quality of high carbon or steel, which is
tempered at the tip. Hardness of knife is essential to obtain good tissue sections.
Most steel knives have been replaces with disposable blades
To achieve good sections knife should be very sharp. Knife are sharpened either manually or by the
use of automatic machine.
Honing - This is done to remove nicks and irregularity from the knife-edge.
Coarse and fine honing is done using different abrasives.
Stropping - The purpose of stropping is to remove the “burr” formed during honing and to polish
cutting edge.
Other types of knives are diamond and glass knives. These knives are very expensive and used for
ultramicrotomy.
Disposable knife – Nowadays these microtome blades are used. Two types of disposable blades are
available.
1. Low profile blade - Usually used for cutting small and soft biopsies like kidney and liver biopsies.
2. High profile blade-Used for any tissue like myometrium, breast tumor or skin.
Advantages
1. No sharpening, honing or stropping the knife.
2. Resistant to both corrosion and heat.
3. Hardness of blade can be compared with the steel knife.
Disadvantages
1. Relatively expensive
2. Disposable blades are not as rigid as steel knife:

Care of the Microtome Knife

Store the knife in its box, when not in use.
The knife should be cleaned with xylene before and after use.
When knife is being stored for a long time, it should be smeared with grease or good grade of light
oil.
Knife edge should not be touched.
Knife edge should never be come badly nicked. It is advisable to use separate knife for cutting hard
issue like bone. The above points are important if re usable knife is being used.