Microtomy for paraffin and frozen sections

Introduction

Microtomy is the means by which tissue is sectioned and attached to the surface of a glass slide for further microscopic examination. The basic principles are applicable to both paraffin and frozen sections although most microtomy is performed on paraffin wax-embedded tissue blocks.

The instrument used to cut sections is the microtome. This has an advancing mechanism which moves the object, the paraffin block, for a predetermined distance until it is in contact with the cutting tool, the knife or blade. The specimen moves vertically past this cutting surface and a tissue section is produced.

This chapter will discuss the techniques necessary to provide quality slides for microscopy in clinical and research histology. Good technique takes practice.

Types of microtome

There are several types of microtome, each designed for a specific purpose, although many have multifunctional roles.

Rotary microtome

This is often referred to as the “Minot” after its inventor. The basic mechanism requires the rotation of a fine advance hand-wheel by 360° degrees, moving the specimen vertically past the cutting surface and returning it to the starting position. The rotary microtome may be manual, i.e. completely manipulated by the operator; semi-automated with one motor to advance either the fine or coarse hand-wheel; or fully automated, when two motors drive the fine and the coarse advance hand-wheel. The mechanism for block advancement may be retracting or non-retracting. Its advantages include the ability to cut thin 2–3 μm sections and its easy adaption to all types of tissue sectioning. Technological advances in the automation of microtomy have improved section quality, increased productivity and improved occupational safety for the technologist. Eliminating manual hand-wheel operation of the microtome reduces the incidence of repetitive motion disorders, a common occupational health problem in the histology laboratory.

Base sledge microtome

Here the specimen is held stationary and the knife slides across the top of it during sectioning. Used primarily for large blocks, hard tissues or whole mounts, it is especially useful in neurological and ophthalmic pathology but 3 μm sections are difficult to produce. Further information regarding sectioning of undecalcifed bone is available in Chapter 17 .

Rotary rocking microtome

Commonly used in cryostats, the retracting action moves the tissue block away from the knife on the upstroke, producing a flat face to the tissue block.

Sliding microtome

The knife or blade is stationary and the specimen slides under it during sectioning. This microtome was developed for use with celloidin-embedded tissue blocks used primarily for research.

Ultra-microtome

Used exclusively for electron microscopy, see Chapter 21 .

Microtome knives

There are many shapes, sizes and materials for microtome knives. Knives were developed to fit specific types of microtome and to cope with different degrees of hardness of tissues and embedding media. Most steel knives have been replaced with disposable blades, although exceptions include the tool-edge knives for resin and steel knives for some cryostats.

Disposable blades

These have revolutionized microtomy. Disposable blades are used for routine microtomy and cryotomy, providing a sharp cutting edge which can produce almost flawless 2–4 μm sections. Disposable blade holders are incorporated into the microtome or an adaptor may be purchased. The blades may be purchased in dispensers, with or without a polytetrafluoroethylene (PTFE) coating which allows ribbons to be sectioned with ease. Note:

The clearance angle should be adjusted in small increments to eliminate problems which occur with ribboning of the tissue. Low profile blades have a clearance angle of 3-5 ^o , high profile 5-7 ^o .
Over-tightening the disposable blade in the clamping device may cause cutting artifacts, e.g. thick and thin sections.
The clamping device must be clean and free of defects.
During sectioning the hand-wheel must be turned slowly.
Extremely hard tissues may pose a problem for disposable blades.

Reliability of a constant sharp edge, ease of use, low or high profiles adaptable to a variety of tissue and paraffin types, and low cost relative to steel knife sharpening, make these blades the mainstay in most laboratories.

Glass and diamond knives

Glass and diamond knives are used in electron microscopy and with plastic resin-embedded blocks.

Paraffin section cutting

Equipment required

Flotation (water) bath.
Slide drying oven or hot plate.
Fine pointed or curved forceps.
Sable or camel haired brush.
Scalpel.
Slide rack.
Clean slides.
Teasing needle.
Ice tray or cooling platform.
Chemical-resistant pencil or pen.
Electronic slide labeling instrument.

Flotation (water) bath

A thermostatically controlled water bath is used for floating out tissue ribbons after sectioning. The temperature of the water in the bath should be 10°C below the melting point of the paraffin wax to be sectioned. Care should be taken to prevent water bubbles from being trapped under the section and this can be accomplished by using distilled water in the bath. Alcohol or a small drop of detergent may be added to the water to reduce the surface tension allowing the section to flatten out with ease.

Drying oven or hot plate

Drying ovens incorporate fans which keep the warm air circulating around the slides. The temperature setting should be approximately that of the melting point of the paraffin wax. If the oven is too hot there may be distortion to the cells causing dark pyknotic nuclei, nuclear bubbling and loss of nuclear detail. Drying times vary depending on the type of tissue, the number of slides to be dried and size of the drying device. Many automated stainers have drying ovens as part of the instrument and the time and temperature is easily regulated. Special care should be taken when drying delicate or central nervous system tissue, a lower temperature is required to prevent splitting and cracking of the section and 37°C for 24 hours is recommended. Time for drying slides should be monitored as many immunohistochemical or special stains are heat sensitive.

Brush and forceps

These or teasing needles are helpful in removing folds, creases and bubbles which may form during floating out of the section on the water bath. They are also helpful for manipulating the section as it passes across the edge of the blade.

Slides

For normal routine work, 76 × 25 mm slides are universally used. Although slides are available in a variety of thicknesses, 1.0–1.2 mm thickness are preferred because they do not break easily. Most slide racks are made to accommodate this slide size. Larger slides are available for use with specialty tissues such as eyes or brains. Unique identification numbers or codes, patient name or other information should be etched, embossed or written on each slide. Automated instruments which imprint the patient’s information on the glass slide are available. Chemical-resistant pens and pencils are routinely used to label the slide. Slides which are positively charged or pre-treated with an adhesive resist detachment of the tissue from the slide during staining. Colored, frost-ended slides may be used for specialized techniques.

Section adhesives

Providing clean slides are used and sections are adequately dried, the problem of sections detaching from the slide during staining should not occur. Occasions when sections may detach from the slide are:

Exposure to strong alkali solutions during staining.
Cryostat sections for immunofluorescence, immunohistochemistry or intraoperative consultation.
Central nervous system (CNS) tissues.
Sections which are submitted to extreme temperatures.
Tissues containing blood and mucus.
Decalcified tissues.

Adhesives may alleviate the problem of tissue loss. Protein adhesives such as albumen, gelatin and starch may be prone to bacterial growth or heavy staining but close monitoring will prevent these problems. Other adhesives which may be used are:

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