Modern microtomes are precision instruments designed to cut uniformly thin sections of a variety of materials for detailed microscopic examination.² For light microscopy, where magnifications can reach up to 1,800x, the thickness of a section can vary between 1 and 10 microns (thin sections). For electron microscopy, where magnifications of several hundred thousands are possible, the thickness of a section is usually of the order of 10 nanometres (ultra-thin sections).

All microtomes consist of three main parts:

• Base (microtome body)
  
• Knife attachment and knife
  
• Material or tissue holder
  

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

Types of microtome and their uses
Microtome styles of manufacture vary depending upon the application required.

ROTARY MICROTOME (Fig 1)
Machines of this sort are general purpose microtomes for cutting semi-thin to thin sections for light microscopy. The microtome operation is based upon the rotary action of a hand wheel activating the advancement of a block towards a rigidly held knife. The block moves up and down in a vertical plane in relation to the knife and therefore cuts flat sections. Available machines range from lightweight, rotary microtomes suitable for cutting paraffin wax embedded material in a continuous ribbon to heavy duty, motor driven instruments used with a slow, continuous speed and retracting advance movement to section hard material embedded in synthetic resin. The rotary microtome can also be found in most cryostats for cutting frozen sections.

Section thickness settings range from 0.5µm to 60µm on most machines. Sections of paraffin wax embedded tissues are normally cut within the range 3 to 5µm whilst resin sections are between 0.5 to 1µm. Rotary microtomes are especially suited to cutting sections using disposable steel knives.

SLEDGE MICROTOME (Fig 2)
These are designed for cutting large blocks of paraffin and resin embedded material including whole organs, for light microscopy. The knife holding clamps allow the knife to be offset to the direction of cut, a major advantage when sectioning large, hard blocks. The microtome, which is very heavy for stability and not usually subject to vibration, can also be used to cut materials from various industrial applications (wood, plastics, textile fibres). They are not suitable for cutting very hard resins such as araldite because of the risk of vibration.

FREEZING MICROTOME (Fig 3)
This form of microtome is used for cutting thin to semi-thin sections of fresh, frozen tissue and semi-thin sections from industrial products such as some textiles, paper, leather, soft plastics, rubber, powders, pastes and food products. The freezing microtome is equipped with a stage upon which tissue can be quickly frozen using either liquid carbon dioxide, from a cylinder, or a low temperature recirculating coolant. Some cooling systems also allow the knife to be cooled at the same time. The cutting action of the freezing microtome differs from those described previously as in this case the knife is moved whilst the tissue block remains static. The block moves by a pre-set amount, in microns, at the end of each cut. Consistent, high quality, thin sections are very difficult to obtain with this type of microtome.

ULTRAMICROTOME (Fig 4)
The ultramicrotome is used to prepare ultrathin sections for light and electron microscopy. Very small samples of tissue or industrial product are usually embedded in hard resin before cutting. It has been reported that sections can be cut as thin as 10 nanometres.³

Two forms of advance mechanism have been developed in this style of microtome. The thermal mechanism relies upon heat induced expansion in a bifurcated metal strip whereas in the mechanical form a microprocessor coupled to a precise stepping motor controls the advance mechanism.⁴ The cutting stroke is motor driven to provide a regular, smooth motion for sections of even thickness and constant reproducibility. Knives are usually made from glass, diamond or sapphire. The block is brought to the knife edge under microscopical control and as each section is cut it is floated on to a water bath adjacent to the knife edge.

CRYOSTAT (Fig 5)
A cryostat is primarily used for cutting sections of frozen tissue as well as pastes, powders and some food substances. The cryostat commonly consists of a microtome contained within a refrigerated chamber, the temperature of which can be maintained at a preset level. A recent innovation has the body of the microtome positioned outside the refrigerated chamber. The cryostat usually contains a rotary microtome although some portable units utilise a rocking microtome. With the object, object holder and knife all at the same temperature and all other conditions for cutting the material optimal, sections as thin as 1 micron are possible.

SAW MICROTOME
Saw microtomes will cut sections from very hard material such as undecalcified bone, glass or ceramics. The samples, commonly embedded in resins, are moved extremely slowly against a diamond coated saw rotating at approximately 600 rpm. Sections of 20 µm or greater are possible providing the saw blade is in perfect condition. Very thin sections are not possible.

VIBRATING MICROTOME
Originally conceived as a microtome which could produce high quality sections of fresh, unfixed material from animal or botanical sources and to replace the hand microtome. The name of the instrument derives from the high speed vibration produced in a safety razor blade to provide the cutting power. The amplitude of vibration is adjusted by altering electrical voltage applied to the 'knife'. Different degrees of vibration are required to produce sections from varying densities of material. To prevent tearing, soft material is cut whilst immersed in a fluid which also aids in dissipating heat produced at the vibrating edge of the razor as it cuts.

ROCKING MICROTOME
Produced in large numbers early in the 20th century, the rocking microtome, comprising three moving parts, is extremely reliable. Designed only for cutting paraffin sections the tissue moves through an arc as it advances towards the knife (the slightly biconcave heiffor knife is used) which is held rigid causing the sections to be cut in a curved plane. Very thin sections are difficult to obtain and one major disadvantage is a limit to the size of block which can be cut. Because of the lightness of the frame the microtome has a tendency to move during cutting. The rocking microtome has largely been replaced by the more precise rotary microtome although it is re-appearing in portable cryostats. Now mainly used for botanical applications sections of 5 µm in thickness are possible.

HAND MICROTOME
The successful use of a hand microtome is limited to sectioning intrinsically rigid botanical material. It is difficult to obtain thin, even sections of animal tissues.

Microtome knives
STEEL KNIVES
Steel microtome knives are manufactured from high quality carbon or tool grade steel which is heat treated to harden the edge.⁵ The steel should be free from impurities, contain anti-corrosives and be rust-resistant. The best knives are those that are fully hardened. Those which are only surface hardened lose the cutting edge very quickly once the hardened area is removed through repeated re-sharpening.

NON-CORROSIVE KNIVES FOR CRYOSTATS
These are manufactured from hardened, heat treated stainless steel free from all impurities and containing 12 to 15% chromium.

DISPOSABLE BLADES
Disposable microtome blades are essentially refined, thickened razor blades. When held in a specially adapted knife holder the blades consistently produce high quality sections and have replaced conventional microtome knives in many instances. All disposable blades are manufactured from high quality stainless steel although there are different grades according to the thickness of the blade. The edge of disposable blades can be coated with platinum⁶ or chromium⁷ to enhance strength and prolong cutting life. Teflon coated blades are particularly suitable for use in cryostats as these offer reduced cutting resistance and minimal friction. The smaller, thinner disposable blade also reaches cryostat chamber temperature more rapidly than a conventional knife minimising time delay during blade exchanges or temperature adjustments.

Disposable blades need to be held rigid in a special holder to prevent vibration during the cutting stroke. These knives consistently produce high quality sections virtually free from compression.

TUNGSTEN CARBIDE
Knives manufactured from high quality tungsten carbide are non corrosive, practically non magnetic and 100 times harder than hardened tool steel.⁸ The knives have excellent resistance to wear but are brittle because of their extreme hardness and should be handled carefully. Up to 30,000 serial sections of undecalcified bone embedded in methacrylate per sharpening has been reported.⁹

GLASS KNIVES
The cutting edge of glass knives used for conventional sectioning is parallel to one surface of the glass ('Ralph knives' with edges of 25 or 38 mm) whilst in those used for ultramicrotomy it is across the thickness of the glass. A commercial glass knifemaker is recommended to ensure consistency and reproducibility of the knife edge. Different profiles of 'Ralph knife' for cutting sections from different embedding media (Fig 6) can be produced very quickly.

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

DIAMOND KNIVES
Diamond knives are manufactured from gem quality diamonds without flaws.^10-11 Although this makes them very expensive the knives are extremely durable, because of the hardness factor of the diamond, and are used primarily for cutting very thin, resin sections.

SAPPHIRE KNIVES
These knives are manufactured from one piece of solid sapphire artificially produced from an alumina monocrystal under computer controlled thermal conditions.⁷ Sapphire is harder than tungsten carbide or glass which ensures high durability of the cutting edge for all types of material. The only restriction when using a sapphire knife is block size as the knife edge is limited to 11 mm. A special knife holder is required.

Profile of steel knives
Microtome knives of hardened steel are made to four different profiles for cutting various materials (Fig 7).¹¹

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

Profile B: plano concave
This knife is similar to a profile A knife but has a thicker back. It is used for cutting sections from material which is too hard to cut with a profile A knife but can also be used for softer materials embedded in paraffin wax. This profile knife is also suitable for cutting the softer components (stalks, leaves) of fresh botanical specimens. This knife should be positioned obliquely to the material being sectioned.

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

Profile D: plane Shaped
This knife will cut hard and tough material as it has greater stability than any of the other profile knives. As only one bevel provides the cutting edge this knife is the least sharp of all of the profiles. It is commonly used for cutting synthetic resin blocks, hard materials embedded in paraffin wax, large wax blocks and various substances used in industry.

The cutting edge of all steel knives is produced by grinding a bevel on each side of the knife for profiles A, B and C, or onto the angled surface of a profile D knife. The bevel faces enclose a sharper angle than the main surfaces of the knife (Fig 8).

Sharpening steel knives
A sharp knife edge free from imperfections is essential for the production of good sections. However, with the introduction of disposable knives the practice of sharpening traditional microtome knives has all but disappeared. Yet the need for a more stable cutting edge occasionally arises and it becomes necessary to sharpen a solid knife.

This can be achieved manually or by using an automatic knife sharpening machine. Automatic machines tend to remove more metal during sharpening so that knives become worn quickly. Manual methods on the other hand remove far less metal but require more skill, experience and time to produce a satisfactory edge.

A sharp edge can be restored quickly to a knife with judicious manual stropping without the need for coarse sharpening provided the knife edge is not damaged. Otherwise coarse cutting compounds should be used initially to remove the imperfections.

Fine honing and stropping is best done manually using diamond lapping compounds. Coarse sharpening can be performed manually or by using automatic knife sharpening machines.

COARSE KNIFE SHARPENING
Automatic
In most instances the knife is held rigid whilst being moved against a rotating drum, rotating wheels or a lapping plate. Abrasive compounds of various grades, from coarse to fine, move against the knife edge. The knife blade is automatically turned at preset intervals so that each side is evenly sharpened.

Lubricants are essential for successful knife sharpening. These agents cool the knife edge (heat generated by the abrasive procedure can destroy the 'temper' of the steel) and continually remove the fine metal particles, produced by the abrasive process, away from the knife edge (see later).

Manual
Coarse honing can also be performed manually using a lapping stick coated with diamond paste containing industrial diamonds with sizes up to 25 µm diameter.¹²

Lapping sticks made from hard woods are used for coarser honing whilst a soft wood is better for finer honing and stropping.

FINE KNIFE SHARPENING
Fine honing is achieved by applying diamond paste, containing industrial diamonds of 1 µm or less, to a lapping stick which is then moved against the knife edge. For manual honing and stropping a knife bevel must be fitted to the back of the knife to ensure the correct angle of bevel for the edge of the knife.¹²

STROPPING
Stropping polishes the knife edge and removes fine metal burrs retained along the edge after honing. Leather, coated with a fine rouge powder or a lapping stick coated with a diamond paste containing industrial diamonds of less than 0.5 µm are effective for this purpose. It is important that very little pressure is used when stropping.

MANUAL METHOD FOR COARSE OR FINE SHARPENING AND STROPPING
Microtome knives are extremely sharp and must always be treated with due care and particularly during manual sharpening. A simple distraction can result in severe injury. The simplest manual sharpening method is that using diamond paste. These contain diamonds of a known size and enable the operator to remove minute quantities of metal and obtain a finish to a knife edge which is almost impossible to achieve using any other form of technology.

MATERIALS REQUIRED
1 Each knife must have its own stropping bevel which is essential to maintain the correct cutting angle to the knife edge.
2 A vice (or other device) to hold the knife firmly during the sharpening procedure.
3 Diamond paste containing industrial diamonds of 1µm diameter for stropping and finishing.
4 Diamond paste containing industrial diamonds of 6µm diameter for fine sharpening.
5 Diamond paste containing industrial diamonds of 14µm diameter for coarse sharpening.
6 Hardwood and softwood lapping sticks measuring 15 x 2 x 0.75 cm.
7 Aerosol can of lapping fluid.

METHOD
1 The stropping bevel is placed along the back edge of the knife to ensure the correct bevel to the edge of a knife is produced.
2 The knife is clamped in position in a holding device firmly held by its handle.
3 A small quantity of diamond paste is rubbed evenly over the surface of a lapping stick. (During the sharpening procedure diamonds become embedded in the wood of the lapping stick, particularly the softwood sticks used for fine sharpening and stropping, so that in time paste need only be applied sparingly.
4 After coating with diamond paste the lapping stick is firmly placed against the side of the knife with the stick touching both the edge of the knife and the stropping bevel.
5 The lapping stick is then moved along the knife in a filing action with continuous, even pressure maintained throughout the stroke. Each side of the knife must be given the same number of strokes. Fifteen strokes on each side should be sufficient to sharpen a blunt but undamaged edge whereas thirty strokes are required for stropping (Fig 9).

Other manual sharpening methods use natural stones, slabs of stone or glass plates as a supporting surface for a lubricant and an abrasive compound.

Lubricants are essential with these methods. It is best to use a non aqueous lubricant (thin lubricating oil) in order to prevent the knife edge from rusting. If an aqueous lubricant is used the knife should be thoroughly cleaned after the sharpening and coated with a fine film of oil. Soap, detergent, glycerol and water soluble oils have all been used as lubricants.

Abrasive powders various agents act as abrasives including carborundum (silicon carbide), alumina (aluminium Oxide), magnesium oxide, chromium oxide and iron oxide (rouge).

Abrasive powders are supplied in a variety of grade sizes. Coarse powders are effective for restoring damaged edges whilst finer powders are used for polishing and stropping.

Microtomy in the biological laboratory
Section cutting
There are many factors which affect the production of good sections. Some of these are:

Fixation and embedding: Animal and human tissues are too soft when fresh to be cut thinly. Some form of pre-treatment is required to harden the tissue to facilitate cutting thin sections. This consists of either freezing or embedding tissues in a medium which offers support for cutting.¹³

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

The correct clearance angle: Necessary to prevent compression in cut sections. The correct clearance angle is also important to reduce friction as the knife edge passes through the block. The clearance angle is that between the knife edge bevel and the block (Fig 8). Various angles have been recommended^14-15 with between 2 and 4 degrees for paraffin sections and between 5 and 7 degrees for resin or frozen sections being most effective. Determining the exact angle is largely a matter of trial and error.

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

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

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

Microtomy in the industrial laboratory
Microtomy in the industrial laboratory covers a wide range of natural and synthetic products and is used to produce specimens for examination by various optical procedures such as fluorescence, incident light darkground, incident light phase contrast, incident light interference contrast and incident light brightfield. Thickness of the cut section can range from ultra thin to thick, depending upon the type of material and the embedding agent used. In industry where there is a more direct relationship between the two an awareness of the nature of the material under investigation and how it will react with chemical agents, solvents, dyes and cutting forces is essential.

The methods of processing and cutting are largely determined by the nature of the specimen, with a satisfactory result achieved more by trial and error than by any systematic approach. Among the embedding agents used, synthetic resins give the best results provided the material under investigation is compatible with the resin. Some specimens require dry cutting without embedding.

Textiles
These includes fibres such as wool, cotton, silk and nylons. Since the nature of textile fibres differs widely there are no standard methods for embedding and cutting. Each set of fibres must be evaluated on an individual basis and a suitable method selected.² Textile fibres are usually cut in cross section to observe and determine structure and the depth of penetration of any dyes used. When fibres need to be cut longitudinally resin embedding is recommended as this holds the fibres more firmly.

Methods available for embedding samples utilise paraffin wax, resins or a combination of paraffin wax and resin, depending upon the fibre type, the need for serial sections and the thickness of section required (see Methods for Industrial Applications). Rotary microtomes with a wedge knife are suitable for cutting most fibres embedded in wax or wax mixtures. A base sledge microtome is more suitable for cutting resin sections with a wedge knife set obliquely in the cutting plane.

Rubber
Rubber samples should be fixed firmly on to a specimen block with a suitable adhesive, such as araldite then frozen to -80°C before cutting. Cooling is achieved by placing the sample and specimen block into a container immersed in a mixture of solid carbon dioxide and acetone.

Rubber sections can be very difficult to cut despite the low temperature. Use a wedge shaped knife inside a cryostat or a base sledge microtome with a wedge shaped knife set obliquely in the cutting plane. Knives can be cooled using a thermocouple. Once removed from the freezing mixture rubber heats and expands quickly and thus should be sectioned without delay. Collect sections with a fine camel haired brush.

Once collected sections should be dipped in a small amount of 70% ethanol then floated on to warm water to flatten out (caused by a difference in surface tension). Mount the section immediately on a clean glass slide and allow to dry on a warm hotplate.

Alternatively the section can be placed on a clean glass slide and expanded with a drop of xylene. If the process is performed while viewed under a stereo microscope, the creases can be gently removed with a fine, camel haired brush. Once flat, a coverslip is placed over the section and the edges sealed with nail varnish.

Woods and wood mixtures
Sections of 3 to 5 µm can be obtained from most woods after embedding in resins using a 7:3 mixture of butyl:methyl methacrylates. Pressed boards and plywood can be cut dry (see Methods for Industrial Applications). Wood should be cut on a base sledge microtome with a wedge shaped knife set obliquely in the cutting plane.

METHOD FOR SOFT WOODS (NOT EMBEDDED)
The sample should be restricted to a maximum face size of 2 cm x 2 cm. Prepare a clean, smooth surface and paint with a liquid, plastic polymer. This will infiltrate through the wood which should be dry enough to cut in 20 to 30 minutes at room temperature. With a sharp, wedge knife sections of 10 to 20 µm should be possible. Thinner sections can be obtained if the subsequent method for hard wood is used.

METHOD FOR HARD WOODS (not embedded)
Sections of hard wood will cut at approximately 10 to 20 µm, depending upon the hardness of the material and the sharpness of the knife.
METHOD
Maximum block face size 2 cm x 2 cm.
1 Boil the wood in distilled water until the wood submerges (this can take up to a few hours for very hard woods).
2 Dehydrate with 70% ethanol using at least 3 changes over a period of 24 hours.
3 Transfer to a solution of glycerin/70% ethanol and impregnate with at least 3 changes of solution over a period of 24 hours.

SECTION CUTTING
A solid microtome, such as a base sledge, is best suited to cutting sections of wood because of its stability and capacity to hold the knife at an oblique angle. A wedge knife should be used. Sections are easier to obtain from wood impregnated with alcohol/glycerin, if the surface of the wood is kept wet with glycerin or 70% ethanol. Sections tend to curl on cutting and can be flattened with a soft, camel haired brush. Place the section on to a clean glass slide and mount under a coverslip using canada balsam as a mounting medium.

Paper, film and foils
The best sections for light microscopy are obtained using the resin method described in the section Methods for Industrial Applications.

The following method is faster but produces a section of inferior quality. The microtome of choice is the base sledge incorporating a wedge shaped knife set obliquely in the cutting plane.

METHOD
1 The sample to be cut is covered on each side with a piece of plastic adhesive film (Sellotape or similar).
2 Place the paper, with adhesive film, between two plastic plates. This allows the material to be held firmly in the microtome clamps. The plastic plate should be of a material soft enough to be cut.
3 Once the block has been trimmed flat a piece of adhesive film is placed over the cut surface. The procedure then follows that described for dry cutting in the section Methods for Industrial Applications.

Leather
Sections of leather for light microscopy can be prepared using the dry cutting, or preferably the resin method described in Methods for Industrial Applications. A rotary microtome can be used but the base sledge offers more rigidity.

Plastics
Hard plastics can be sectioned using the dry cutting method described in Methods for Industrial Applications. The microtome of choice is a base sledge microtome with a wedge knife set obliquely in the cutting plane. Soft plastics are difficult to cut with this method because they tend to bend freely. These are best frozen to -20°C using solid carbon dioxide then cut in a cryostat or on a base sledge microtome using a cooled wedge knife. If a base sledge is used the specimen must be kept frozen.

Plastic foams
Plastic foams are not suited to embedding techniques because of the air spaces trapped within the material and because agents used in embedding can dissolve soft foams. If the foam is firm it can be cut using a base sledge microtome with a strongly plano-concave knife although the sections tend to be thick. Thinner sections can be obtained by freezing the foam to -20°C with solid carbon dioxide then using a cryostat with a wedge shaped knife. Alternatively a base sledge microtome fitted with a wedge knife cooled by a thermocouple can be used provided the specimen is kept frozen.

Powders and pastes
Powders for sectioning are either loose or compacted.

Loose powders: These need to be embedded. Methacrylate is the method of choice as the crystalline nature of most powders allows them to be pulled from a wax embedded specimen during cutting. Sections are prepared using a base sledge or rotary microtome with a wedge shaped knife.

Compacted powders: These can be cemented on to a microtome block and cut using the dry cutting method. An alternative is to slowly infiltrate the compacted powder with a plastic monomer, polymerise and section using a base sledge or rotary microtome.

Pastes: Because pastes contain moisture they are best frozen with solid carbon dioxide then sectioned in a cryostat using a wedge shaped knife.

Pigments and pastes
The method for sectioning pigments and paints will depend upon the nature of the specimen. Provided that the paint or varnish does not dissolve in the solvents wax, wax/resin or resin embedding methods can be used. Flakes of hardened material can be cut using the dry cutting method.

Food products
The nature of the food product will largely determine the sectioning procedure. Hard, dry materials such as seeds and husks are best embedded in methacrylate. Other food products, such as fresh and processed meats can be processed and sectioned using standard histological methods. Pastries are frozen and cut in a cryostat.

Methods for industrial applications
Dry cutting
This technique can be used for a wide range of materials including, wood, paper, leather, some plastics, varnishes, pigments and metals. Sections obtained always curl or fray during the dry cutting procedure and it is advisable to use an adhesive polymer or adhesive foil to keep the specimen together.

METHOD
1 After trimming to a flat surface, a piece of adhesive tape is pressed on to the surface of the block before each section is cut.
2 The section, attached to the adhesive, can be examined directly or both can be mounted under a coverslip using a suitable mounting medium.

Wax method
Suitable for wool, cotton silk, some rayons and most foodstuffs.

REAGENT REQUIRED
Mix together in a heated crucible:
Paraffin wax (melting point 58°C) 9 parts
Beeswax 0.5 parts
Carnauba Wax (vegetable wax) 0.5 parts<

METHOD
1 Pour the hot wax mixture over a small piece of the specimen (block face size 1 cm x 1 cm) positioned in an embedding mould or fibres fixed firmly in an embedding frame as shown in figure 10. (The embedding frame should be placed on a flat surface such as a glass plate so that hot wax does not flow from the underside). The slots in the frame, in which the fibres lie, are sealed with adhesive tape before pouring the embedding mixture into the mould.
2 Allow the wax to solidify at room temperature. Avoid rapid cooling as this may encourage the development of cracks in the hardened block.
3 Trim the block with a sharp blade then cut sections in the normal way using a wedge profile knife. Float sections on to warm water to flatten and collect onto a clean, glass slide.
4 Remove the wax from the section with xylene then mount the fibres under a coverslip using canada balsam as a mounting medium.

Resin and wax method
This procedure is suitable producing ribbons of serial sections (5 to 6 µm) of all types of fibres except for those that are very hard or incompatible with the resin (such as polyamide and polyacryl nitrile). Paper, leather, pigments and foodstuffs can also be prepared this way.

REAGENT REQUIRED
Mix together in a heated crucible:
Candelilla wax 5 to 6 parts
Carnauba wax 1 part
Beeswax 1 part
Colophony 1.5 parts
Venetian turpentine 1.5 parts

METHOD
1 Pour the hot resin/wax mixture over the dry specimen in an embedding mould or fibres mounted in a metal frame (Fig 10) which has been placed in a desiccator. Evacuate the air carefully to ensure that the embedding medium does not foam. Cool in air and trim to size with a heated knife.
2 Allow the block to set for approximately 2 hours then cut and float sections on to warm water to flatten.
3 Collect sections on to clean, glass slides and allow to dry thoroughly.
4 Remove the resin/wax mixture by treating with hot trichlorethylene or carbon tetrachloride. Both of these substances are extremely hazardous and this procedure should always be performed under a suitable fume extraction unit. Protective clothing comprising a long sleeved laboratory coat or gown with elasticised wrist bands, rubber gloves, safety goggles and a respirator, with canisters suitable for the chemical fumes should be worn.
5 Mount the section under a coverslip using canada balsam as a mounting medium.

Resin method for light microscopy
This method is applicable to all types of specimens including textile fibres, natural fibres, woods, paper, leather, plastics, paints and pigments. A rotary microtome fitted with a glass knife or a base sledge microtome with a wedge shaped knife set obliquely in the cutting plane are best used for cutting resin sections.

The resin is a mixture of methyl and butyl methacrylate. Stabilisers (usually hydroquinone) normally incorporated into methacrylate resin to prevent polymerisation during transportation or storage, must be removed before embedding.

REMOVAL OF RESIN STABILISER
METHOD
1 Pour the resin to be cleared into a separating funnel of appropriate size.
2 Add (approximately 10% v/v) a 5% to 10% aqueous solution of sodium hydroxide.
3 Shake vigorously for 1 minute. A brown deposit forms and settles to the bottom of the separating funnel after which it can be drained.
4 Repeat the procedure until the sodium hydroxide remains clear.
5 The methacrylate becomes cloudy after this procedure. It can be clarified by repeating the procedure using distilled water.

REAGENT REQUIRED
RESIN EMBEDDING MIXTURE
The exact proportions will vary depending upon the type and number fibres to be embedded² (Table 1).

Between 1% and 2% by volume of accelerator (normally N-N dimethyl aniline) is added to the volume of resin to initiate polymerisation.

METHOD
1 Gelatin capsules are suitable as embedding moulds - fibres can be drawn through the top and bottom of the capsule using a suture needle (Fig 11) or material for embedding can be processed in situ in the capsule.
2 Tie a knot in the fibres at the bottom end of the capsule.
3 Pour the polymer resin into the capsule.
4 Place the capsule lid over the base and pull the fibres tight. Polymerise at 48°C for 5 to 10 hours. Polymerisation at temperatures above 48°C can cause bubbles to form in the methacrylate.
5 The hardened block is removed by immersing the capsule in water.
6 Trim the block with a razor blade and cut sections using a plane wedge knife or glass knife.
7 Sections can be removed from the knife edge with a camel hair brush, transferred to warm water to flatten and then collected on to clean glass slides.
8 Mount sections under a coverslip using castor oil as a mounting medium. The edges of the coverslip need to be sealed with nail varnish to prevent evaporation of the mounting medium.

Principles of section cutting
The basis of all good sectioning is a combination of experience and a well prepared knife edge.

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

The objective is to produce a ribbon of artefact free, flat sections from which one to several are selected and mounted on to clean slides.¹⁵ Common problems that occur in section cutting are listed in Table 2.

METHOD
1 Set the blocks on to a cold surface to harden the face to be cut (a refrigerated cold plate or ice). Avoid prolonged cooling and very cold surfaces as both can cracks the surface of the paraffin wax block.
2 Install a sharp, trimming knife in the microtome and set the correct clearance angle, normally 2 to 5^o. (See Knife and cutting angles).
3 Trim a paraffin wax block, with a sharp blade, so that the sides are parallel and 2 to 3 mm of wax surrounds the tissue.
4 Fit the trimmed block into the block holder and orientate so the edge offering least resistance meets the knife edge first (Fig 12).
5 Advance the block until it just touches the knife edge.
6 Coarse cut the block at 15 µm until the full face has been trimmed.
7 Return the trimmed block to the cold surface for 1 to 2 minutes.
8 Set the advance feed to the desired thickness (3 to 5 µm for most purposes).
9 Remove any debris associated with coarse cutting from the knife edge with alcohol. (Xylene should not be used as it often leaves an oily remnant on the knife to which cut sections will stick).
10 Install a fresh, sharp knife in the microtome or move the previous knife to a new, unused area.
11 Re-install the cold block in the microtome and cut a series (or ribbon) of sections at the required thickness. Gently breathing upon the sections as each is cut dissipates static electricity, flattens the section and facilitates movement of the ribbon down the knife. Section compression is minimised by using a sharp knife set to the correct clearance angle.
12 The ribbon is separated from the knife edge with a moist camel hair brush and pulled across the surface of a warm water bath (Fig 13). (Section expansion will compensate for the compression caused when cutting).

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

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

Section adhesives
Providing the section is sufficiently dried it will adhere to the slide adequately for most staining procedures. Alkaline solutions, however do tend to remove sections from glass slides and in these instances an adhesive will be necessary.

Sections from some tissues, for example those containing high concentrations of blood (blood clot, spleen, bone marrow), nervous tissue (brain, spinal cord), skin and decalcified tissues, are also prone to detaching from slides. The routine use of a section adhesive for these tissues is also recommended.

Poly-L-lysine
Poly-L-lysine has excellent adhesive properties without the background staining that occurs with many other adhesives. It is particularly useful for sections to be stained with immunoperoxidase methods. Poly-L-lysine is available as a solution or solid in the hydrobromide form.

REAGENT REQUIRED
Solution
Diluted 1 in 10 with distilled water before use.

METHOD
1 Immerse clean glass slides. Immerse in poly-L-lysine solution for 10 minutes at room temperature.
2 Drain then dry the slides at 60°C or overnight at room temperature before use. Cover the slides so that dust is not attracted to the surface during the drying process. Once dried the slides can be stored in the original slide box until required.
3 The surface should be moistened to reactivate the poly-L-lysine before mounting sections.

Albumin
Although Mayer's original method used fresh egg white, other sources of albumin work equally well. A disadvantage is that albumin tends to absorb dyes causing background staining.

REAGENT REQUIRED
Mayer's Egg Albumin¹⁶
Fresh egg white 50 ml
Glycerol 50 ml
Mix thoroughly, using a magnetic stirrer, then add a crystal of thymol or 1 gm of sodium salicylate as a preservative.

METHOD
A small drop of egg albumin smeared on the slide before the section is mounted and dried is a suitable section adhesive.

Celloidin
Celloidin will take up colour if aldehyde fuchsin, Schiff's reagent, mucicarmine or alcian blue are used. In other respects it is a strong adhesive and is especially effective for decalcified material.

REAGENT REQUIRED
Prepare as a 0.5 to 1% solution in ether/alcohol.

METHOD
1 Deparaffinise the slide in xylene then wash in absolute ethanol.
2 Coat the slide with the celloidin solution by brief immersion or by dropping the solution from a dropper bottle.
3 Allow the slide to air dry at room temperature for up to five minutes.
4 Harden the coating by immersing the slide in 80% ethanol for 1 to 2 minutes.
5 Rinse with distilled water.

Gelatin
Gelatin is superior to egg albumin as an adhesive but will also give background staining. It can be applied to the slide in a small drop and smeared over the surface or can be added to the water flotation bath. Solutions from 0.5 to 5% gelatin in distilled water, have been used with success but require a preservative (bactericide and fungicide). If gelatin is used in a water flotation bath the solution should be changed daily to prevent the growth of organisms.

If firmer adhesion is required, gelatin can be treated with formalin vapour which renders the gel irreversible. A coplin jar, with a tightly fitting lid, containing a few millilitres of concentrated formalin can be used (in a fume hood) for this purpose. Ensure that the section does not contact the liquid formalin and leave for one hour at room temperature. After treatment the slide is dried on a hot plate in the normal manner.

Chrome - Gelatin (suitable for decalcified bone sections)
REAGENT REQUIRED
Chrome - gelatin reagent
Gelatin 1.5 g
Chromic potassium sulphate 0.5 g
Distilled water 100 ml
Mix well then filter before use.

METHOD
1 Slides are loaded into racks, washed thoroughly in hot, soapy water then rinsed in at least two changes of distilled water.
2 Dip the slides in the chrome - gelatin solution for a few seconds, remove, cover and allow to dry at room temperature. If the slides are not covered dust can settle in the adhesive medium.

TECHNICAL NOTE
Coated slides will store for many months without deterioration. Albumin is a suitable substitute for gelatin in the above formula.

Methyl Cellulose
Methyl cellulose prepared as a 0.5 to 1% solution in ether/alcohol can be used in place of celloidin.

METHOD
1 Deparaffinise the slide in xylene then wash in absolute ethanol.
2 Coat the slide with the methyl cellulose solution by brief immersion or by dropping the solution from a dropper bottle.
3 Air dry at room temperature for up to five minutes.
4 Harden the coating by immersing the slide in 80% ethanol for 1 to 2 minutes.
5 Rinse with distilled water.

Resin
A 10% solution of araldite in acetone, smeared on to slides immediately before use, is not affected by dyes and solvents once polymerised. It may be necessary to extend staining times when resin adhesive is used although in some cases staining is more pronounced.¹⁴

Starch
Starch being a carbohydrate should not be applied to slides when carbohydrates are the substance under investigation. It is a stronger adhesive than egg albumin or gelatin alone having similar properties to those of gelatin followed by formalin vapour.

REAGENT REQUIRED¹⁷
Starch 3 gm
Cold distilled water 30 ml
Mix into a paste then add 60 ml of boiling distilled water. Mix then add 0.5 ml of concentrated hydrochloric acid. Continue boiling for a further five minutes. A crystal of thymol is added after cooling as a preservative.

METHOD
A small drop is smeared along the slide before mounting the section.

Sodium silicate
Sodium silicate lightly etches the surface of glass slides enhancing section adhesion. It has the disadvantages of retaining silver giving a black background when silver methods are used and also stains red with the methyl green pyronin technique.¹⁴ Sodium silicate is used as a 10% aqueous solution.

RESIN SECTIONING
Glass or steel knives are used for cutting resin embedded material. The choice of knife will depend upon the type of material to be cut and the size of the block. For instance a glass knife is recommended where sections under 2 µm are required whereas for sections of undecalcified bone a tungsten carbide knife is required.¹⁸

For most purposes a motorised microtome with a retracting specimen holder provides the best sectioning results for resin embedded material. The motor provides an even cutting speed which is difficult to duplicate with manual operation. The retracting specimen holder prevents the block face from meeting the back of the knife on the upstroke which would damage both the knife edge and the block face.

METHOD
1 Trim the block leaving 3 to 4 mm of clear resin on the leading edge of the block (that which first meets the knife edge) and 1 to 2 mm of clear resin on the trailing edge. Trim the sides of the block to within 0.5 to 1 mm of the tissue as an excess of resin on the sides may cause wrinkling.
2 Fit the trimmed block into the block holder on the microtome. Sections are usually cut through the longitudinal axis of the block to reduce mechanical stress when the block meets the knife edge (Fig 15).
3 Advance the block until it just touches the knife edge.
4 Coarse cut the block, at a maximum of 5 µm (more than this and both the block and knife edge can be irreparably damaged) until the full block face has been cut and a representative area of tissue exposed.
5 Sections can be cut wet (preferred for epoxy resin) or dry.

For wet sectioning attach a water trough to the knife edge or use a wet cotton swab.¹⁹ Position the tip of a wet cotton bud on the edge of the knife. As the section is cut it is transferred to the cotton bud with a slow anticlockwise roll of the cotton bud.

Section adhesives are generally not required but should be considered for undecalcified bone sections.

RESIN REMOVAL
Hard resin provides a stubborn barrier to most stains and is best removed before staining.²⁰ Methacrylate cannot be removed.

Of the many agents for resin removal before staining, sodium or potassium ethoxide are simple to prepare and easy to use.²¹

REAGENT REQUIRED
Prepare a solution of potassium or sodium hydroxide saturated in absolute ethanol. Store in a tightly stoppered container until the solution turns brown (usually 2 to 3 days). Filter before use.

METHOD
1 Remove the resin section from the hotplate and immerse in a coplin jar of filtered reagent for 2 to 3 minutes.
2 Rinse well in absolute ethanol.
3 Rinse in 95% ethanol.
4 Wash in distilled water.

Acknowledgments
I wish to thank Mr. Grant Garwood, Photographer at The Queen Elizabeth Hospital, Adelaide, Australia for the black and white photography

References
1 Lillie RD. Histologic technic and practical histochemistry. New York; McGraw-Hill Book Company. 1947

2 Walter F. The microtome manual of the technique of preparation and of section cutting. Germany; Ernst Leitz Wetzlar GMBH. 1980

3 Du pont. Sorvall JB-4 Microtome. The light microtome for super thin sections. Newtown, USA; Du Pont Instruments. 1991

4 Du Pont. Sorvall MT-5000 Ultramicrotome. Wilmington, USA; Du Pont Biomedical Products Division. 1991

5 Glen Creston. Glen Creston microtome knives. Glen Creston Ltd. Stanmore, Middlesex, U.K. 1985

6 Feather Industries. Feather disposable microtome; Technical data. Feather Industries Ltd. Tokyo, Japan. 1987

7 Histoline. Blades for all occasions. Histoline Inc. California. 1987

8 Austral Scientific. Disposable tungsten carbide microtome blades and holder for paraffin and frozen sectioning. Canberra ACT; Austral Scientific. 1986

9 Wild Leitz. Characteristics of Tungsten Carbide Knife AS-STC 170D. Wild Leitz Australia Pty. Ltd. 1988

10 Diatome. The diamond knife for light microscopy. Diatome Ltd. Switzerland. 1987

11 Diatome. The Swiss diamond knife. Diatome Ltd. Switzerland. 1987

12 Ellis RC. Diamond Paste Knife Sharpening. Aust-J-Med-Tech 1971; 2: 24-27

13 Culling CFA. Handbook of Histopathological Technique. London; Butterworths. 1957

14 Gordon K; Bradbury P. Microtomy and Paraffin sections. In Bancroft JD and Stevens A ed. Theory and Practice of Histological Techniques, 3rd edition. London; Churchill Livingstone. 1990

15 Luna LG. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology 3rd edition. Washington DC. McGraw-Hill. 1968

16 Baker FJ; Silverton RE; Luckock ED. An introduction to Medical Laboratory Technology, 2nd edition. London; Butterworths. 1957

17 McDowell AM; Vassos GA. Comparison of starch paste and albumin mixture as agents for routine mounting of paraffin sections. Arch Pathol 1940; 29: 432.

18 Gormley BM; Smith R. Resin Embedding Workshop Notes. Australian Institute of Medical Laboratory Scientists, (SA Branch). Flinders Medical Centre, South Australia. 1982

19 Chew S. A method for cutting resin sections for histology. Tissue Talk 1982; 2:, 3

20 Chew S; Cole K; Watson I. Application of Resin Embedding for Diagnostic Histopathology. Histopathology Workshop, Joint Committee for Continuing Education, Australian Institute of Medical Laboratory Scientists and Western Australian Institute of Technology, Perth, Western Australia. 1982

21 Lane BP; Europa DL. Differential staining of ultra thin sections of epon-embedded tissues for light microscopy. J Histochem Cytochem 1965; 13: 579-582