Cutting of Sections

Sectioning is a process whereby tissues are cut into uniformly thin slices or sections with the aid of a machine, to facilitate the studies under the microscope. The machine or instrument used for cutting sections is known as microtome.

The microtome consists of three essential parts, namely:

1.   Block holder – where the tissue is held in position.

2. Knife carrier and knife – for actual cutting of tissue sections.

3.   Pawl, ratchet feed wheel and adjustment screws – to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections.

Whatever the type of microtome is used, the principle remains essentially the same, that is, a spring–balanced teeth or pawl is brought into contact with, and turns a ratchet feed wheel connected to a microtome screw, which in turn rotated, moving the tissue block at a predetermined distance towards the knife for cutting sections at uniform thickness.

Types of microtome:

1.   Rocking microtome – for cutting serial sections of large blocks of paraffin embedded tissues.

This was invented by Paldwell Trefall in 1881, the simplest among the different types of microtome, consisting of heavy base and two arms – the lower arm resting in pivots and a supporting column, and attached to the micrometer screw, at the base of which is found the Ratchet Wheel with feed mechanism. The upper arm, carrying the block holder on one end by means of a screw, is connected to a lever by a piece of nylon thread.

When the lever is pulled forward, the pawl is brought in contact with the Ratchet Wheel to which the millhead screw is attached. The Ratchet Wheel is turned, rotating the microtome screw. The lower arm is elevated, which in turn raises the upper arm at its fulcrum, thereby carrying the chuck or block holder forward, towards the knife. As the pressure on the operating handle or lever is released, the tension on the spring causes the upper arm to return to its normal position; in an arc of circle, a section is thereby cut as the tissue passes to the knife edge in a slightly curved plane, in 10 – 12u thickness.

The Cambridge rocking microtome, available in two sizes, may be used to cut small and large blocks of paraffin tissues. It is theoretically not recommended for serial sections since tissues are cut in slightly curved planes. It has, however, proved to be ideal for serial sections in actual practice, producing ribbons of 60 – 90 sections with case.

The rocking microtome has recently been incorporated into cryostat for cutting unfixed tissues at a temperature of –20^oC, due to the relatively inexpensive cost of both knife and machine, and the simplicity of the mechanism.

2.   Rotary (Minot) microtome – for cutting paraffin embedded sections

This was invented by Minot in 1885 – 86 to cut paraffin embedded tissues, and is the most common type used for both routine and research laboratories at present. The microtome is operated by rotation of the flywheel, causing reciprocal motion of the knife over the block, the thickness of the section being automatically regulated by the ratchet feed wheel. The pawl is used to come in contact with ratchet wheel which in turn rotates the micrometer screws, thus making the block move towards the knife at a predetermined rate. Adjustment screws have been so that the block is parallel to the microtome knife at all planes.

It is different from the rocking microtome in that the knife and the blockholder are brought together by upward and vertical motions, cutting sections in a perfectly flat plane, thereby allowing excellent serial sections to be cut. It is heavier and more stable than the rocking microtome, is more complex in design and construction, and is therefore more expensive. It may be used for cutting large blocks of tissues although results are better when the sliding microtome is used. The knife is placed in a blade–up position and is therefore relatively dangerous.

Models are now available for cutting ultrathin sections and for cryostat use.

3.   Sliding microtome – for cutting celloidin embedded sections

This was developed by Adams in 1789. There are two types of this microtome. One model, the BASEL SLEDGE MICROTOME, consists of two movable pillars holding the adjustable knife clamps, allowing the knife to be set at an angle for cutting celloidin sections. The chuck or block holder is set on a heavy metal base which can be moved backward and forward under the knife.

The chuck is fitted into a micrometer screw, which in turn connected to the ratchet wheel. When the operating handle is turned, the pawl becomes in contact with ratchet feed wheel, which in turn rotates the micrometer screws. The block holder is then raised towards the knife at a thickness pre–determined by a micrometer gauge.

Such a machine is suited for sectioning specimens embedded in all forms of media, especially for cutting sections from tough tissue block which may offer great resistance to the knife. Larger sections are more easily cute with the knife, set at an angle due to less resistance offered by the block. Sections are cut in a perfectly flat plane, thereby making excellent serial tissue sections. It is comparatively more stable than the ordinary sliding microtome.

The second type, the STANDARD SLIDING MICROTOME, is different from the base sledge microtome because with this instrument, the block remains stationary while the knife is moved backwards and forward during the process of sectioning.

In both of these machines, the knife can be set obliquely for celloidin sections, or straight for large refractory paraffin blocks, cutting both large and small tissues with ease; it is especially recommended for cutting extremely hard and rough tissue blocks.

It is the most dangerous type of microtome due to the movable exposed knife. A slow but very steady motion is therefore required to manipulate the instrument.

4.   Freezing microtome – for cutting unembedded frozen sections.

This was invented by Queckett in 1848. The stage for the block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe thru which Carbon Dioxide passes from the cylinder. A simple lever operated valve allows the release of rapid, intermittent burst of Carbon dioxide which will freeze the block holder and the tissue evenly. A second cooling device for lowering the temperature of the knife is also incorporated in most machines to facilitate sectioning.

The knife holder is attached to a lever which in turn connected to the pawl. When the operating handle is moved back, the knife is moved back to its original position, away from the block. The lever, is in turn moved causing the pawl to get in contact with the ratchet feed wheel and thereby the micrometer screw. The blockholder is then raised towards the knife at a pre–determined thickness. By pulling the operating handle forward, section is cut at the knife edge slices thru the raised tissue block.

The microtome is firmly clamped on the edge of the bench for use, or mounted on especially constructed shelf, with CO₂ cylinder below. It is used to cut undehydrated tissues in a frozen state, especially in instances when rapid diagnosis is required, when histological demonstration of fat is needed, when certain neurological structures are to be studied, and when sensitive tissue constituents to be studied are damage or destroyed by heat.

The Cryostat or Cold Microtome

The cryostat is an apparatus used in fresh tissue microtomy, which is capable of freezing the tissue into the blockholder to the correct degree of hardness to facilitate easier and faster sectioning. It consists of a microtome, kept inside a cold chamber which has been maintained at a temperature between –5^oC to –30^oC (average is –20^oC) by an adjustable thermostat, capable of freezing fresh tissue within 2 – 3 minutes and cutting sections of 4u with ease. The usual microtome used is a Cambridge Rocking Microtome.

It provides a means of preparing large, thin, often unwrinkled sections of fresh frozen tissues especially when Fluorescent Antibody Staining Technique or Histochemical enzyme studies are required. It also permits rapid preparation of tissue biopsies for surgical pathology.

5.   Ultrathin microtome – for cutting sections for electron microscopy.

The ultrathin microtome is primarily used for cutting tissue sections at 0.5 micra, for electron microscopy. The knife used for cutting the ultrathin sections consists mainly of selected fragments of broken plate glass. The specimen used is small, fixed Osmium Tetroxide, and embedded in plastic.

Care of the microtome

After sectioning, all the accumulated paraffin and small pieces of tissues must be brushed away with a soft brush and not allowed to stay in the microtome, since this may later on interfere with the cutting of tissue blocks. After carefully drying the machine and knife holder, the parts should be wiped with xylol. Prolonged and continuous application of the painted parts with xylene should, however, be avoided since this reagent is capable of removing the paint. Movable portions should be oiled thoroughly to prevent rusting. The microtome must always be covered when not in use, to prevent accumulation of dust and other dirt which may later on interfere with the normal sectioning of tissues.

Types of tissue sections

1.     Paraffin Sections

Sections are usually cut between 4 – 6u in thickness for routine histologic procedure, after the block has been fixed and secured to the blockholder. The micrometer gauge is set to the required thickness and the knife is positioned in such a way that the center of the blade is in line with the block and the knife has been securely clamped in place.

Cutting is then started until complete sections come out of the block, and a regular cutting rhythm is maintained. The cutting rate depends upon the type of the tissue, the size of the block, and the model or type of the microtome that is used. Sections usually from ribbons due to slight heat generated between the block and the knife edge during the process of cutting.

The knife is usually tilted at 0 – 15”angulation on a microtome to allow a clearance angle between the cutting facet and the tissue block. Biconcave knives require smaller clearance angles than wedge–shaped knives.

Incomplete sections are discarded. Complete ribbons are picked up at once with a camel hair brush, a pair of forceps or the fingers. Tissues, which tend to crumble (e.g. blood clots, bone marrow, spleen) can be sectioned with ease, by exhaling gently into the section while the block is being cut slowly, to reduce the effects of static electricity.

Sections are removed in ribbons of ten, to allow easy location of serial sections. The sections are then floated out on a water bath set at 45 – 50^oC, approximately 6 – 10^oC lower than the melting point of the wax used for embedding the tissue. This is to flatten the sections and prepare them for mounting into the slider. Fold and crease may be removed by stretching the sections gently with a pair of dissecting needles. Bubbles may be teased out from beneath the sections by means of the same needle.

A section is selected for staining and picked up onto a clean slide in a vertical position. The slide is immersed in the water bat in a near vertical position as close as possible to the section. When the slide touches the section, it is lifted vertically out of water and drained. The mounted section is then placed in a paraffin oven to dry.

Sections may also be flattened out by placing them on a slide which has been flooded with 20% alcohol, producing convection currents which will serve to remove the creases in the tissue within a few seconds. Alternatively, sections may be floated out and flattened with a few drops of distilled water on the slide which is later warmed on a hot plate and drained.

Besides the paraffin oven which is maintained at a temperature of 2 – 5^oC above the melting point of the paraffin used, small thermostatically controlled incubators may be used, regulated at 37^oC and at 45 – 55^oC, for enzyme digestion, chemical extraction, metallic impregnation and enzyme localization techniques. Staining of serial sections should never be attempted unless they are completely dried.

2.     Celloidin Sections

Celloidin sections are usually between 10 – 15u in thickness. The blocks are trimmed in the same manner as in paraffin blocks, but they do not require hardening by chilling before cutting. The sections are usually cut with the sliding microtome.

To avoid dehydration and shrinkage, sections are usually cut by the wet method, both the sections and the block being kept moist with 70% alcohol during cutting. Sections tend to roll up during cutting and moistening the block and section with alcohol by means of a camel hair brush will serve to flatten the sections on the knife.

Celloidin sections do not come off in ribbons and have to be collected into 70% Alcohol immediately. They are the stored in the same solution in jars with tightly fitting lids, and finally mounted on to slides after they have been stained.

3.   Frozen sections

Fresh, completely unfixed tissues, or tissues that have been briefly treated with formalin may not require embedding anymore; but instead, may be frozen with carbon dioxide and cut in a freezing microtome or cryostat. Two methods of preparing frozen sections may be resorted to:

a.    Cold Knife procedure

Almost any microtome can be utilized for the purpose, provided means are made available for freezing and maintaining the specimen and the knife at low temperatures, usually by utilizing the carbon dioxide technique.

A piece of filter paper soaked in gum syrup is placed on the microtome stage, and short bursts of CO₂ are applied, freezing the filter paper to the stage. The selected block of tissue, approximately 3 – 5 mm thick, is then oriented on the stage, applied with a few drops of gum syrup, and frozen solid with several intermittent bursts of CO₂, each for 1 – 2 seconds duration, at intervals of around 5 seconds. It should be frozen just to the point where it will be firm enough to section. The tissue is then lifted up to the knife manually and trimmed until the surface is flat. The surface is then warmed with the finger until the hard frozen tissue starts to thaw, and becomes visible to the naked eye. This is the Dew Line, the point at which sections may then be cut at 10 micra thickness.

Sections do not form ribbons but rather stick to the knife blade and should, therefore, be removed with a Camel hair brush or finger moistened with water. They are then transferred to a dish of distilled water to separate and picked up individually for mounting and staining. The water dish is usually placed on a dark or black background, in order to see the sections which are usually colorless or very light in color.

Tissues that have been frozen too hard will usually chip into fragments when cut. The surface of the block may then be softened by warming slightly with the ball of the finger or thumb. Tissues that have not been sufficiently frozen will cut thick and crumble, and the block may come away from the stage. More bursts of CO₂ gas should then be given to refreeze the block.

Whether used in a cold environment or not, a different temperature between the tissue and the knife is usually employed, the latter being colder. Using a cold knife in a controlled cold environment, optimum condition for sectioning shall be provided for by the following temperatures:

            Knife                          –          40^o to –60^oC

            Tissue                        –          5^o to –10^oC

Environment            –          0^o to –10^oC

Success of the procedure depends upon ambient temperature and humidity. It is very hard, if not possible, to cute sections in a hot or humid surrounding. Facility for cutting and quality of sections are always improved if a cold room is utilized.

Sections thinner than 6u generally cannot be obtained even from tissues that section well, and with ideal conditions for sectioning.

b.   Cryostat procedure

This method makes use of the Cryostat, an apparatus used in Fresh Tissue Microtomy, consisting of an insulated microtome housed in an electrically driven refrigerated chamber maintained at room temperature near –20^oC, where microtome, knife, specimen and atmosphere are kept at the same temperature. Majority of the sections can thus be cut in isothermic conditions, where the temperature for sectioning can be accurately established and controlled; accuracy and consistency is thereby guaranteed.

Certain tissues such as fat or mucin, and hard or dense structures in a soft matrix require much lower temperatures to impart a suitable consistency for cutting. These are sectioned on the Cryostat by lowering the tissue or knife temperature or both, either by placing the blockholder in a bath of alcohol or acetone containing dry ice, or by exposing the tissue to carbon dioxide.

The frozen tissue is mounted on the microtome. Both the microtome knife and the tissue block are left in the cryostat for 15 – 30 minutes at –20^oC, to ensure that they are cooled to the correct temperature. Sections between 5 – 10 micra are then cut slowly and steadily, removed from the knife with a camel hair brush, attached directly to slides of cover–glasses at room temperature, air – dried, fixed (optional) and stained.