MICROTOMES AND THAIR TYPES

MICROTOMES 

1. Sliding microtome

2. Base sledge microtome

3. Cambridge rocking microtome

4. Rotary microtome

5. Freezing microtome

6. Cryostat 

Ultra-microtom

MICROTOME KNIVES

(a) Planoconcave profile

(b) Wedge-Shaped

(c) Biconcave Profile

(d) Tool-Edge Profile

TERMSUSED IN MICROTOMYY

Tilt of the microtome knife

Compression

Rate of cutting

Ambient temperature

Orientation

Slant of the knife

• SHARPENING OF MICROTOME KNIVES

• Hand Sharpening

• 1. Belgian yellow stone
• 2. The Belgian black vein (blue-green)
• 3. Arkansas
• 4. Aloxite

Procedure of Honing

• Plate glass honing
• Knife Sharpening Machines (Automatic Hones)
• Plastic blades
• Factory grinding

STROPPING

MICROTOMES

The earliest recorded form of microtomes was a non-mechanical, hand operated device which was nothing more than an elaborated scalpel. The first truly mechanical microtome was perhaps the sliding microtome devised by Adams in 1798. Microtomes are classified into various types, but all of them should fulfil the following requirements:

(a) Rigid support for the knife and the tissue block. 

(b) Means of moving either the tissue block across the fixed knife-edge or the knife edge across the block. 

(c) Means of accurately advancing the tissues to cut each section at the predetermined thickness. 

The choice of microtome depends on the type of work, the nature of the tissue preparation and embedding. Certain types are adapted for special work.

MICROTOMY

Having fixed, processed and embedded the tissue, the next stage is to cut sections from the block. The machines or instruments used to cut thin sections are called microtomes.

The description of the following microtomes are limited to their general features while a prospective buyer is advised to consult the manufacturers for fuller details. 

1. Sliding microtome (Fig. 5.1) This instrument is most useful in the cutting of tissues embedded in celloidin. Unlike the other types, the block remains stationary while the microtome knife moves during the process of cutting.

2. Base sledge microtome (Fig. 5.2) This is a highly rigid and precision made instrument. The tissue block holder is attached to a sledge resting on precision machined runners.

The sledge is pushed beneath a fixed knife. The size and range of adjustment for cutting angles makes it most effective for cutting very hard tissues, and is also useful for dealing with celloidin or LVN embedded tissues by means of a special extended clamp that gives very oblique knife angles. Large tissue blocks up to approximately 8 x 10 cm can be cut when special large tissue stages are attached to the instrument. It can also be adapted for cutting frozen sections. The base sledge microtome is very versatile and so very ideal for a laboratory handling a wide variety of tissues and block sizes.

3. Cambridge rocking microtome (Fig. 5.3) The instrument was invented by Coldwell and Treefall in 1881 and was developed by the Cambridge Company. The name of the microtome was derived from the rocking action of the upper arm. It is a simple machine in which the tissue block is swung across the fixed knife-edge in an arc. The movement is governed by a spring that is located by a handle attached to a piece of cord. After each cutting stroke, the movement of the handle advances the tissue block. Sections are cut in a slightly curved plane and its freed mechanism is graduated in units of 1 or 2 um. Though primarily meant for paraffin wax embedded tissues, it is easily adapted for frozen sections. This is mainly due to the simplicity of its mechanism and small number of moving parts. This machine is however no longer manufactured.

4. Rotary microtome (Fig. 5.4) The rotary microtome was invented independently by the Americans, Charles Minal and Feifer. The instrument is most ideal for routine and research work. It is excellent for cutting serial sections. The microtome is smaller in size than the sledge microtome and has a smaller knife area which limits the size of tissue blocks to about 3 x 4 cm.

The tissue block is moved up and down in a flat plane across the knife-edge. The movement is effected by the turning of a handle at the side of the instrument which simultaneously advances the block after each section is cut.

5. Freezing microtome (Fig. 5.5) The freezing microtome generally consists of a central advancing screw on which the block holder is situated. A cylinder of CO2, the cooling agent, is connected by means of a reinforced flexible lead to the stage which is hollow with perforations around the perimeter. These perforations, known as baffles, allow the gas to flow and easily escape, thus providing uniform freezing of the tissue. To facilitate section cutting, the knife edge is kept cool by means of another cooling device. The more recent models of freezing microtomes are connected to a thermoelectric cooling units called thermal modules instead of CO,. 

The module works on the principles of the Peltier effect. Peltier in 1884 found that by passing an electric current through two different conductors there was either absorption or generation of heat at the point where the two met, depending on the direction of the current. The adaptation of the Peltier effect to the microtome knife stage was by loffle in 1958 though it was only in 1964 that Rutherford and his colleagues fully adapted the thermo-electric cooling device to both knife holders and block stage. With thermal module

there is a continuous thawing and freezing of tissue. The freezing microtome has its greatest application when (1) urgent diagnosis is required, (2) fat is to be demonstrated histologically (3) in the absence of a cryostat (4) when enzymes and neurological structures are to be demonstrated.

It should be noted that with a freezing microtome, serial sections cannot be cut; structural details are somehow distorted; staining of frozen section of unfixed tissue is not very satisfactory and freezing artefacts such as ice crystals in the tissue, ballooning and vacuolation may be introduced.

6. Cryostat (Fig. 5.6) Before the advent of cryostat, the preparation of frozen sections was confined to freezing microtomes which were operated at room temperature with CO2 as the tissueRefrigerant. This method has now been replaced by the cryostat.

The cryostat is a precision machine which is housed in a deep freeze cabinet maintained at a temperature of about -15°C to -30°C. The major advantage of the cryostat is that it maintains the tissue block, knife and the section at the same temperature. This eliminates difficulties such as cutting of unfixed tissues with knife freezing attachment, and obtaining thin sections. Further more there is no more thawing and freezing of the tissue and knife.

The first cryostat was produced in Denmark by Linderstrom-Lang and Morgensen. It was employed in the study of histochemistry in 1938. At first, the cabinet was kept cold by using blocks of dry ice; this was later replaced by refrigerated coils

in 1948. The cryostat was modified by Adamstone and Tailor using a Lietz base sledge microtome in refrigerated cabinet where the knife was kept cold by dry ice being held onto it by troughs on both sides of the cutting area of the knife. The cryostat enjoys the same advantages as the freezing microtome.

 The big disadvantage was the atmospheric conditions which caused rolling up of the sections. For this reason it was originally suggested that the instrument be placed in a cold room. Later on the anti-roll plate and an environmentally controlled refrigerated cabinet were introduced.

The initial freezing or quenching techniques can be carried out by any of the following:

1. Use of cold metal blocks cooled by refrigerated coils. 

2. CO2 expansion coolers. 

3. The use of dry ice applied directly onto holder (snap freezing). 

4. Thermo-electrically cooled holders.

5. Liquid gases, e.g., nitrogen. This is considered the safest and popular way.

Rapid freezing is essential with mammalian tissues if enzyme histochemistry is to be performed. The different models incorporate various types of microtomes. Pearce for one, regarded the Cambridge rocker to be the most suited for use in cryostats because the knife was insensitive to cold of any degree likely to be attained in any cryostat chamber. Pearce also found that the coating for the anti-roll plate with polytetra fluoroethylene or teflon was advantageous. Today anti-roll plates are made of perspex instead of glass. Models of cryostat available include the bench top, free standing, manual and automatic.

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Ultra-microtome

This microtome is a highly specialised precision instrument (Fig. 5.7). It is used for the preparation of ultra thin sections for electron microscopy. It differs from the other microtomes in the following ways:

(i) The block folding mechanism is precision made and the adjustment which is very fine is activated by mechanically or electrically controlled thermal expansion.

(ii) Specially constructed knives of plate glass are used for cutting small blocks not bigger than 1 sq mm embedded in synthetic resin (araldite).

(iii) It makes use of a low power binocular microscope to aid the orientation of the blocks and section cutting.

(iv) Sections as thin as 60-70 nm are cut with ease.

There are other specialised microtomes designed for special purposes. For example, the Jung is designed to cut very hard biological materials such as undecalcified heads of femur.

MICROTOME KNIVES

Microtome knives are available in various sizes and blade profiles (cross-sections). They are classified based on their blade profiles (Fig. 5.8), namely: (a) Planoconcave, (b) Wedge-shaped, (c) Biconcave and d) Tool edge profiles. The length is chosen according to the particular microtome that is in use. Most of them have detachable handle at one end.

(a) Planoconcave profile This knife is used with the base sledge microtome to cut all except the toughest tissues embedded in paraffin wax. It is also suitable for celloidin or LVN embedded materials. When in use, the concave surface should face upwards. It is almost completely vibration free and it is easy and quick to sharpen.

(b) Wedge-Shaped This knife cuts almost all paraffin wax sections with the sledge or rotary 

microtomes. It can be used in both freezing microtome and cryostat fitted with microtomes other than the Cambridge rocker. Because of its shape (plane on both sides), the edge is vibration free and so is able to cut the toughest materials. It takes longer time to sharpen than the planoconcave knife. 

(c) Biconcave Profile It has a hollow ground on both sides. The shape makes this knife confined to the Cambridge rocker microtome. It is also known as the Heiffor blade. It is used mainly for wax-embedded tissues. 

(d) Tool-Edge Profile This type of knife is plane on both sides with a steep cutting edge. It is used on robust microtomes for cutting hard tissues such as undecalcified bone.

Most knives are wedge shaped with the sides inclined at an angle of about 15 degrees. The sur faces are polished so that sections do not stick to them but will move on the surface minimising folding and distortion and thereby facilitating good ribbon formation. The sides of the cutting edge are called cutting facets and the angle formed between the cutting facets where they meet is known as the bevel angle or facet angle and is normally about 270-33o . This angle is kept constant for each knife by means of a slide-on back for use on each knife during honing and stropping. The slide-on back should be pushed in carefully, evenly and symmetrically so that cutting facets are equal. Knives should be inclined relative to the cutting plane so that there is 5-10 degrees clearance between the cutting facet presenting to the block and the surface of the block. Without this clearance the cutting facet will compress the block as it goes under the knife. The effect of this is that there may be no cutting of section at all or very thick and thin sections may be produced.

TERMSUSED IN MICROTOMYY

Tilt of the microtome knife Tilt or the incline of a microtome knife is the angle between the surface of the block and the line which bisects the edge of the knife. For soft blocks, less tilt is used than for hard blocks. There must be sufficient clearance in order to avoid compression of the blocks on passing under the knife and sections being alternatively thick and thin. Tilt angle varies between 10-40 degrees depending on the angle of the cutting facet.

Compression This is the difference which exists between the sides of the section which is cut and the side measurements of the tissue block. The compression of sections can also be influenced by room temperature, the sharpness of the knife and the humidity of the air. Excessive compression causes damage of the cells and tissue structures and it should therefore be kept to a minimum. 

Rate of cutting Soft tissues embedded in soft media, e.g., brain in celloidin, require a slow smooth cutting action. A fast forced cutting movement leads to compression. Blocks embedded in paraffin wax requires a fast stronger movement when ribboning. A good combination of knife, slant, tilt, sharpness and cutting rate is a prerequisite to obtain good sections.

Ambient temperature Paraffin wax gets softened if the temperature is too hot and this presents 

difficulty in obtaining sections. Blocks of ice, preferably in polythene sachets, can be used to cool the cutting surface of the tissue block; prolonged treatment with blocks of iced water should be avoided as it tends to macerate the tissues. In addition, too low a temperature results in the cracking of the surface of the block, variation in thickness of the sections and difficulty in cutting serial sections (ribboning).

Application of nominal degree of heat supplied by breathing on to the surface of the block helps when cutting paraffin section. But a real increase in heat can lead to expansion of the material thereby resulting in thick sections being produced. The degree of heat supplied by breathing onto the block comes with experience.

Orientation Orientation is the proper placement of the blocks on the microtome and this must be in

such a way as to facilitate the cutting of sections of uniform thickness. All microtomes have adjusting mechanisms for the orientation of the blocks in relationship to the knife edge. In practice, most blocks cut well if the blocks are initially trimmed with parallel upper and lower edges and slanting sides. A poor alignment of the paraffin blocks of tissue results in difficulty in cutting single or serial sections.

Slant of the knife For most routine materials, the cutting edge of the knife can be fixed at right angles to the direction of movement of the block. For tough tissues it is more beneficial to present the block to a slanted knife blade. The base sledge microtome allows this kind of adjustment.

• SHARPENING OF MICROTOME KNIVES

There is a cogent need for the microtome knife to be sharp. Successful section cutting to a large extent depends on the microtome knife edge. The histotechnologist should, as a matter of duty, be skilful in handling his microtome knife. When the cutting edge becomes blunt or damaged, the knife should be subjected to sharpening. Sharpening of the microtome knife is divided into two stages: honing or the removal of metal nicks to obtain a straight cutting edge free from nicks, and stropping or polishing the cutting edge. Each of these procedures may be performed either by hand or with the automatic knife sharpener. Before engaging in these procedures, one should be familiar with and be able to identify the various parts of the knife. The heel is the end of the blade closest to the handle, while the other end is referred to as the toe.

The back is a tubular steel device which slides over the back of the knife. It is used when honing or stropping to maintain the bevel of the blade. The bevel is that part of the edge that is in direct contact with the hone. A knife back is not required for a biconcave knife. Each knife must have its own back that corresponds to its size. This is because during honing, the back can also be ground away if longer than the knife. But for stropping, one back can serve several knives.

Hand Sharpening

This can be done with hones or with plate glass and abrasives. The hone is usually a rectangular block of natural or synthetic stone with hard grinding surface for sharpening a knife or other cutting tools. The stone is graded coarse, medium or fine based on the degree of its abrasiveness. The finer the grain in the hone, the harder the hone. The size of the hone used depends on the size of the knife to be sharpened. The hone should be long enough to allow the whole of the knife edge to be sharpened in a single stroke.

Popular stones of various grades of fineness are:

1. The Belgian yellow stone This is a natural stone, is widely used as it gives good results at reasonable speed.

2. The Belgian black vein (blue-green) This stone is equally good.

3. Arkansas This stone like the Belgian stones is a natural one. It is clear white to pale yellow in colour. It is slower than the Belgian stones because it is less abrasive.

4. Aloxite This is a series of composite stones whose abrasiveness ranges from coarse to superfine. For microtome knife sharpening, only the fine and superfine grades are used.

Procedure of Honing

The hone is first wiped clean with a soft cloth to remove loose particles of stone and metal. Wipe with soft cloth moistened in xylene. The hone is then covered with a thin film of lubricant such as soap-water. The knife is fitted with its own back and held obliquely on the stone, edge forward. Gentle even pressure is applied on the knife with thumbs or forefingers, and the knife is drawn obliquely forward on the stone in an easy, steady motion, 'heel' first so that when the 'toe' is reached it is still on the stone. The knife is then turned over on its back, and with the heel leading again, it is still steadily drawn towards the operator. This is called the 'heel' to 'toe' motion (Fig. 5.9a). Such sequence as described makes up a double stroke.

The honing is complete when all the large nicks have been removed and the cutting edge is sharp and straight. The knife is wiped clean with a gauge moistened in xylene. The edge may then be viewed with low power objective of the microscope to ascertain the removal of nicks. Very large nicks require regrinding of the knife edge and this is better done in the factory.

Plate glass honing A piece of plate glass 610 mm inches thick, about 35 cm long and 2-5 cm wider than the knife blade is used. It is used for grinding and removal of nick in conjunction with an abrasive. The abrasive in common use is carborundum 303-304, followed by aluminium oxide or corborundum 305. 

Diamantine is used for the final polishing. The abrasives are suspended in oil or water and applied to the surface of the glass plate. One advantage of the plate glass is that the abrasives come in varying grades of particle sizes, so that all types of honing can be done. Also, since the plate is wider than the length of the knife blade, honing is done by pushing and pulling forward and backward at right angles to the transverse axis of the plate. For sharpening procedures, the particle size must not be larger than 30 um and for polishing, not greater than 8 um.

Knife Sharpening Machines (Automatic Hones)

These machines are fast becoming indispensable in histopathology laboratories. They are time saving and fairly easy to manipulate so that an inexperienced hand can produce a well sharpened knife with an even bevel. There are some that can safely be referred to as semi-automatic while others are fully automatic (Fig. 5.10).

The semi-automatic machines require hand feeding of the knife against wheels made of glass or metals. Lapping compounds made up of suspensions of alumina or diamond grit aid the sharpening process by being continuously recirculated by means of a built in pump. Lapping compounds are supplied in varying grades and the condition of the cutting edge of the knife determines the grade of the lapping compound selected.

One big disadvantage of this type of honing machines is that the manual feeding of the knife across the revolving wheel causes uneven pressure and variation in the rate of honing resulting in uneven knife edge.

Some models of the semi-automatic sharpening machines also have facilities for stropping.

In the fully automatic honing machines the knife is fitted into a holder attached to the main spindle and allows the cutting edge to be in contact with the circular plate made of metal or glass. An adjusting mechanism brings the height of the glass plate to the level of the bevel of the knife. The abrasive is spread evenly on the surface of the plate by oscillatory and rotatory movement of the device. The duration of sharpening time is set depending on the state of the knife; the knife automatically turns over from edge to edge at suitable intervals. The sharpening speed is also set based on the condition of the knife, slow speed being reserved for knives in poor conditions.

Plastic blades These are disposable blades which can be adapted to fit most types of microtomes. The necessity of sharpening is eliminated. Although they are becoming increasingly popular, they are expensive.

Factory grinding When the cutting edge has retreated into thicker metal due to repeated sharpening, it results in the widening of the bevel angle. When this angle becomes much greater than 350, the knife should be returned to the factory so that both its wedge surfaces may be ground down and the correct bevel angle restored.

STROPPING

Following honing the edge of the knife is polished on a leather strop made from the best quality horse hide. The strop surface should be of ideal size, e.g., 7-9 by 45 cm. The strop may be hanging type or one fixed to a solid wooden block. The hanging type strops are attached by one end to a bench or wall at a suitable height for the user. 

The strops are pulled as far as possible to prevent sagging and create enough tension to prevent rounding the cutting edge of the knife. A canvas back is fitted to most of these strops and serves as a preliminary stropping surface before using the leather surface. Stropping is manipulated with one hand while the other hand pulls on the

strop.

 The movement is that of toe to heel', just the reverse of the honing process (Fig. 5.11). The stropping surface is usually impregnated with a fine abrasive (diamond dust or fine carborundum).

The rigid type is preferred by many histotechnologists because it is easier to manipulate and rounding of the knife edge is minimised. Some workers consider stropping unnecessary when honing has been properly carried out. However, stropping a knife after honing or after each session of section cutting definitely enhances the cutting ability of the knife.