EMBEDDING And MICROTOME

EMBEDDING

 5. EMBEDDING

In this stage processed tissue blocks are now externally supported by the same supporting

medium as used in the impregnation stage. Tissues are embedded using supporting medium

in molten state with the help of different moulds. It gives an external support which then

facilitates sectioning on a microtome. Different types of moulds are used for making blocks.

Most commonly used are briefly described below:

5.1 LeXFNKDUW¶V0RXOGV

These are convenient moulds for routine work and are widely used. They comprise of two Lshaped bits of metal, normally metal, and might be bought in various sizes. They are

arranged on a glass or metal plate to forma mould (socket) of the desired size. When a

socket is obtained tissue specimen is put at the base of socket and then molten paraffin wax

is dispensed. The tissue block is pressed with a warm forcep. Then more wax is poured and

block is allowed to cool for solidification. For cooling, blocks can be kept in refrigerator or

on cold plates if available.

Advantages:

i. These moulds give even blocks with parallel sides.

ii. They are adjustable to give a wide variety of block sizes.

On the other hand, they are somewhat cumbersome and too slow for a busy

laboratory.

5.2 Steel Base Moulds

The system using steel base moulds and tissue embedding cassette is given the name

³7LVVXH 7HN 6\VWHP´ 7KH ELRSVy is placed in depression of the base mould and then

paraffin wax is dispensed. The tissue biopsy is properly centered and oriented with warm

forcep. The tissue embedding cassette is applied and wax is poured in its cavity. Then the

steel base mould with fixed embedding cassette is kept on a cold plate for quick, even

solidification of the embedding medium to get a paraffin block. Steel base mould can be

reused whereas embedding cassette gives a permanent paraffin, block and fits in chuk

adapter of microtome. The steel base moulds are pre-coated with a release compound in

alcoholic solution so that the wax does not adhere to them.

 

Advantages:

There are various advantages of the Tissue Tek System. Some of which are:

i. Handling of tissue block is very easy. Embedding cassettes are directly fixed in

block holder (Chuk Adapter) of the microtome.

ii. Embedding cassettes can also be used for processing of tissue biopsy.

iii. Numbering of blocks is very easy; as a separate specified area is present in the

cassette.

iv. Transportation of blocks in the form of embedding caste is very easy. 

5.3 Watch Glasses

These are ideal for embedding fragmentary biopsies. It is not essential to smear them with

glycerol, but it is a sensible precaution because sometimes blocks are difficult to remove.

There are not commonly used in the laboratory

MICROTOME

6. MICROTOME

The magnifying lens is intended to work with the investigation of creature tissue by communicated light and for this reason the tissue should be cut into slight lamellae or segments. To obtain such thin

VHFWLRQVRIXQLIRUPWKLFNQHVVDGHYLFHLVXVHGWHUPHGDV³0LFURWRPH´The first microtome

was invented by George Adams and Alexander Cummings in 1770. Another scientist from

Czech Republic, -DQ(YDQJHOLVWD 3XUN\QČ, who was a physiologist, further developed the

device which was the first to be used practically. 

Figure 6.1 18th Century Microtome by Alexander Cummings

Adapted from Journal of the Royal Microscopical Society, 1910, pages 779-782. The Royal

Microscopical Society, Oxford, England. 

There are different designs of microtomes but all possess some basic requirements. All

microtomes must have a rigid support for tissue block as well as for microtome knife. After

each cutting stroke, advancement of the tissue block towards the knife edge with respect to

predetermined microns. The means of movement of tissue block across a fixed knife or

sometimes movement of knife across a fixed tissue block. The flywheel in many

microtomes can be operated by hand. This enjoys the benefit that a well put together can be made,

as the generally huge mass of the flywheel keeps the example from being quit during

the sample cut. The flywheel in more up to date models is much of the time coordinated inside the microtome

casing.

Different types of microtomes are now available commercially. Some impotent types are

briefly described below:

6.1 Cambridge Rocking Microtome

This microtome is very simple in design. It is not manufactured now a days but remains a

favourite microtome for many workers because of its simplicity of operation and

support and its capacity to create segments of superior grade.

Principle:

In this microtome the tissue block is swung across a fixed knife edge in the form of an arc.

A spring is attached with the operating hand which controls the movements of advancement

as well as of block across the knife. Segments ready on the Cambridge shaking microtome are cut in a slightly curved plane.

The Cambridge shaking microtome was quite possibly the earliest instrument to be integrated into

a cryostat by British manufacturers for the preparation of sections from unfixed tissue at a

temperature of about -20o

C. This was because of its simple mechanism and small number of

moving parts. However, nowadays rotary microtome is used in cryostat.

Figure 6.2 Cambridge Rocking Microtome

Adapted from http://www.radicalscientific.com

Advantages:

i. This microtome is very simple in design and easy to operate.

ii. It is also cheaper and free from high custom duty.

iii. Small and soft tissue blocks can easily be sectioned.

Disadvantages:

i. This microtome is only suitable for small sized blocks (1-2cm2

).

ii. The sections obtained are slightly curved, so not so much uniform in thickness and

therefore disturbs focusing under the microscope.

iii. Tough material cannot be sectioned by this microtome.

6.2 Rotary Microtome

It is the most widely used sectioning device in routine histopathological laboratories. This

was invented by Minot in 1885-1886 and independently by Pfeiffer in 1886. Pfeiffer was a

mechanic at Johns Hopkins.

Principle:

In this microtome the Tissue block moves across a fixed knife edge in a vertical plane, i.e.

by vertical rise and fall movement perpendicular to knife edge. The vertical movement and

advancement movements are controlled by an operating handle which is in the form of a

rotor or wheel on one side of the machine. Due to this shape of operating handle, the rotary

microtome is given this name. The block holder of rotary microtome is equipped with

adjusting screws to ensure that the block is parallel to the microtome knife in all planes.

Figure 6.3 Rotary Microtome

Adapted from http://www.radicalscientific.com

Advantages:

i. The rotary microtome is very useful for serial sectioning.

ii. This microtome is more rigid then Cambridge rocking microtome. 

iii. Sectioning of somewhat tough material is also possible.

iv. Tissues blocks of size 3-4 cm2 can also be sectioned easily.

v. This microtome is used in cryostat, although other types were initially used.

vi. It is recommended for research labs because it gives sections of superior quality. 

vii. It is also recommended for laboratories with high workload because it is easy to

operate and tissues of uniform thickness can be obtained.

Disadvantages:

i) This microtome is complex in design and construction and this complexity leads to its

high cost.

ii) It is not suitable for large blocks or very tough material embedded in celloidin or LVN.

The knife is also dangerously placed (blade up)

6.3 Base sledge Microtome

The base sledge microtome is heavy duty rigidly constructed machine adaptable for

separating examples implanted in all types of media.

Principle:

In this microtome the tissue block resting on a type of sledge is pushed horizontally beneath

a knife edge that can be slanted. So, larger sections can more easily be cut with the knife set

at an angle. By changing the slant angle, less resistance is offered by block in sectioning.

This microtome is excellent for cutting sections from blocks of tough tissue, especially if

the blocks are huge and offer stamped protection from the blade. This microtome may be

adjusted for frozen segment cutting by substitution of the paraffin wax object holder with

either a CO2 freezing stage or a thermo module.

Figure 6.4 Base Sledge Microtome

Adapted from http://websites.labx.com

6.4 Sliding Microtome

The fundamental difference between the sliding microtome and those described on previous

pages is that with this instrument the block remains stationary while the microtome knife

moves during the process of sectioning.

The main value of sliding microtome is the ease with which it cuts sections from tissue

embedded in celloidin. A number of instruments of varying designs are produced

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6.5 Freezing Microtome

Freezing microtome is one of the methods for obtaining frozen sections. It is used primarily

for cutting sections of fixed tissue:

a. When speed is of the utmost importance.

b. When a cryostat is not available.

It is also required to demonstrate fat histologically, and when certain neurological structures

are to be studied.

Principle:

In this microtome representative portion of a biopsy is rapidly frozen in a circular stage.

This stage is perforated at the periphery and is supplied with a cylinder of CO2 under

pressure. As CO2 escapes freely through the perforated stage, lowering of temperature

occurs and tissue block is frozen at the top surface of the stage. Then sectioning is

performed by a movable knife which can move across the surface of the fixed frozen tissue

block.

This is a crude method in which there is no temperature control and one can suffer a lot of

problems during the sectioning. The freezing microtome varies extraordinarily from those

machines used for preparation of paraffin wax sections.

Thermo Modules:

Thermoelectric cooling units may be used in place of CO2 gas to freeze the tissue and cool

the knife. These units referred to refrigeration capacity and function by a phenomenon

NQRZQDVWKH³3HOWLHU´HIIHFW7KHVHXQLWVDUHSURGXFHGFRPPHUFLDOO\DQGDUe designed to

fit a large number of microtomes. But on the other hand they are now less commonly used

because of the widely used cryostat. For freezing microtome, optimum cutting temperature

for the tissue is usually about -20o C.

6.6 Cryostat

The best method of preparing sections from unfixed tissue is by use of a cryostat. The

cryostat consists of a microtome housed in a deep freeze, cabined, maintained at a

temperature of approximately -15 to -30o C. A thermostat controls the temperature of

cabinet. The optimum working temperature of cryostat is -18 to -20o C. The microtome is

handled by on operating handle outside the cabinet.

There is a variety of cryostats manufactured; the fundamental difference between them is

the type of microtome employed. The earliest models manufactured in UK incorporated the

Cambridge rocking microtome. However, nowadays rotary microtome is commonly used.

Several models fitted with base sledge microtomes, suitable for cutting larger and tougher

tissue blocks, are also available. Wedge Profile knife is used in Cryostat necessary. 

Cryostat, are usually provided with a rapid freezing attachment for this purpose and for

attaching tissue blocks to the block holder. This latter is extremely valuable when the

instrument is to be utilized for planning dire segments from biopsies. The tissue blocks not

to be sectioned immediately should be quenched and stored at a temperature of -20o C in

airtight containers or aluminum foils. To keep away from the arrangement of enormous troublesome ice

gems while freezing new tissue, fast freezing (extinguishing) is important. 

The maintenance and procedure of using cryostat is described in chapter 10s. 

6.7 Laser Microtome

Laser Microtome operates using a cutting action of an infra-red laser. It is used for contact

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or chemical fixation, thereby minimizing the artifacts from preparation methods.

6.8 Parts of a Microtome

The basic parts of a microtome are as follows:

ƒ Operating handle, this is rotated to move the tissue block for cutting and advancement

movements

ƒ Tissue block holder, with fixing screws

ƒ Thickness gauge, to set the selected microns

ƒ Knife clumps with screws, for knife support

ƒ Operating handle lock

ƒ Knife safeguard

ƒ Feedback mechanism: It is used to move the block towards or away from the knife edge.

When in operation, it controls the regular advancement movement of the block after

each cutting stroke for the predetermined microns.

6.9 Microtome Knives

The microtome knives are of two main types as follows:

6.9.1 Those which require sharpening and stropping; and

6.9.2 Disposable knives which are actually disposable blades with disposable blade

holder. 

The first class of knives is further classified according to their cross-section (profile) into 4

classes as follows:

6.9.1.1 Wedge Profile Knife:

This knife is wedge shaped in its cross-section, i.e. broader at the base and tapering towards

the edge. This knife is plane on both surfaces. It is rigid because its edge is well supported.

It is widely used for sectioning of tissue blocks embedded in different media. It is also used

for frozen sections in a cryostat. Disadvantage is that during the sharpening a knife back is

required which keep the surfaces of knife apart from the hone.