MICROTOMY – THE ART OF SECTION CUTTING
Microtomy or section cutting is the technique of making the very thin slices of tissue specimens for the microscopic examination to identify the abnormalities or atypical appearance in the tissue (if present) and also for the study of various components of the cells or tissues like Lipids, Enzymes, Antigens or Antibodies (Immunohistochemistry), Cell organelles etc.
Before sections are made the tissues require to be embedded in some fluid, which will permeate their interstices, and is capable of being rendered firm so as to support the most delicate parts when the knife passes through the tissue. The most commonly used embedding medium is Paraffin wax as it allows the optimal sample preparation.
After the process of dehydration and clearing, the tissue is placed in the embedding medium for the infiltration and impregnation, considered together as internal embedding. After which the external embedding is done by placing the internally embedded tissue into a mould and paraffin wax is poured into the surroundings to make a paraffin wax block which is called as external embedding or Block preparation.
This paraffin-embedded tissue block is thus sectioned to obtain the thin slices of the tissue specimen which is further stained using suitable staining techniques and mounted to preserve the stained section for future reference.
Nowadays, the most commonly used microtome is Rotary microtome which allows the perfect sectioning of the paraffin-embedded tissue specimens and serial sections can easily be obtained. Most commonly the section cutting is done at the thickness of 4-6μ (microns).
For the ideal sectioning of the tissue, certain accessories and types of equipment are required apart from the Microtome and paraffin block which are as follows:-
- A pair of pointed forceps
- Small squirrel hair brush
- A sharp scalpel
- Water bath with thermostatic control
- Paper towel/Clean cloth
- Clean microscopic glass slides
- Section adhesive (Mayer’s Albumin, Gelatin)
- Blotting paper and ice cubes
The requirements & procedure for preparing Mayer’s Albumin is as follows:
- Egg Albumin – 50 ml
- Glycerol – 50 ml
- Sodium salicylate – 1gm
- Thymol – 1-2 Crystals
Mix well all the components. Filter the solution thus obtained with the help of cheesecloth and used up to 5-6 months.
Trim the block with the hot knife before beginning the microtomy or section cutting. In a properly trimmed block face, the top and bottom edges will be parallel; the block face should be in touch with the knife. All the tissues desired on the slide should be exposed to the face and no scratch marks should be visible on the surface.
If the scratch marks are visible on the block face, the knife must be moved laterally to use a new portion of the knife edge and the face is trimmed again. Accordingly, the microtome was prepared for the process of section cutting:
- The feed mechanism should be screwed back
- The knife position should be checked
- The indicator for the thickness of the section should be adjusted and kept between 4-6μ.
- If there is enough paraffin in front of the tissue untrimmed, then the block must be cut until the tissue reaches the knife.
The knob attached to the spring holder was moved quickly, so as to obtain a ribbon enabling the tissue to adhere to each other. When the Serial section/Ribbons are obtained they may be transferred, with the help of small squirrel hair brush & forceps, into the tissue floatation bath and the temperature should be maintained below the melting point of the wax.
This will remove any wrinkles (if present in the section) and flattens the sections. The ribbons are then cut and shorten by using a hot pointed forceps into lengths of about two and a half inches and transferred onto the clean grease free glass slide smeared with the adhesive (e.g. Mayer’s Albumin) for the better adhesion of the section so that it will not get washed away during the process of staining.
Several ribbons may be placed side by side, and so a large number of sections kept in the order in which they are cut. A mark should be made on the slide to indicate where the series begins, and each slide should be numbered so that the exact position of each section in the series can be recognized at once.
The slides are kept aside for a few hours to dry and after that, the sections should be stained using suitable staining technique.