TOPIC # 1 HISTOPATH LAB: MICROTOMY

MICROTOMES

● From the Greek mikros, meaning "small", and temnein, meaning "to cut- is a tool
used to cut extremely thin slices of material, known as sections. Important in science,
microtomes are used in microscopy, allowing for the preparation of samples for
observation under transmitted light or electron radiation.

MICROTOMY

● Is a process by which tissue can be sectioned and attached to a surface so that

microscopic examination can take place.

TYPES OF MICROTOMES

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

embedded tissues

2. Rotary microtome - for cutting paraffin embedded sections.

3. Sliding microtome - for cutting celloidin embedded sections.

4. Freezing microtome -for cutting unembedded frozen sections.

5. Cryostat or cold microtome – for cutting frozen sections

6. Ultrathin microtome - for cutting sections for Electron Microscopy.

MICROTOME KNIVES

TRIMMING AND SECTION-CUTTING ARE DONE WITH A MICROTOME KNIFE, WHICH

IS AVAILABLE IN THREE BASIC TYPES OR SHAPES:
 1. Plane-Concave Knife (usually 25 mm. in length) - One side of the knife is flat while
the other is concave. Less concave sides are recommended for cutting
celloidin-embedded tissue blocks on a sliding microtome. More concave sides are
used to cut paraffin sections on base-sledge, rotary or rocking microtome.

2. Biconcave Knife (usually 120 mm. in length) - with both sides concave,
recommended for cutting paraffin - embedded sections on a rotary microtome

3. Plane-Wedge Knife (usually 100 mm. in length) have both sides straight,
recommended for frozen sections or for cutting extremely hard and tough specimens
embedded in paraffin blocks, using a base sledge type or sliding microtome.

Plane-wedge and plane-concave knives- The angle formed between the cutting
edges is known as the "Bevel Angle", normally about 27° to 32°.

The perfect and optimum cutting angle is obtained when the sides of the wedge knife
are inclined at an angle of about 15°, causing maximum penetration of the tissues
and minimizing distortion.

To prevent uneven sections, or alternate thin and thick sections, the knife should be
inclined with a 5-10° clearance angle from the cutting plane so that the cutting facet
will not compress the block during the process of cutting.

HONING AND STROPPING

HONING

● Involves the removal of gross nicks on the knife edge (Coarse Honing) to remove
blemishes, and grinding the cutting edge of the knife on a stone (Honing Proper) to
acquire an even edge.

● This procedure makes use of a hone, a natural sharpening stone or hard grinding
surface (carborundum), which serves to remove nicks and irregularities on the knife
edges

SEVERAL TYPES OF HONES MAY BE USED:

Belgium Yellow - for manual sharpening when cutting edge has been rendered blunt
or nicked. This type usually gives the best result.

Arkansas - gives more polishing effect than the Belgium Yellow.

Fine carborundum - is much coarser than the first two types and is used only for
badly nicked knives followed by either one of the first two knife sharpeners.

● The surface of the hone is wiped clean with a soft cloth moistened with xylene in
order to remove the scattered small particles of stones and metal. It is then covered
 with a thin film of Mineral and Clove Oil, Xylene, Liquid Paraffin or Soapy Water for
lubrication.

● Mechanical honing with machines may make use of a vibrating frosted glass plate or
a wheel driven by an electrical motor. The knife is pressed against the flat side of a
rotating glass wheel which is being driven by a mechanical device.

STROPPING

● Stropping is the process whereby the "burr" formed during honing is removed and the
cutting edge of the knife is polished.

● The purpose of stropping is to polish and sharpen the cutting edge, while that of
honing is to remove the irregularities from the knife.

● Around 40- 120 double strokes are usually required.

TYPE OF KNIVES

1. GLASS KNIVES

● Glass knives are generally used for trimming and semi-thin sectioning of tissue
blocks for electron microscopy. They are prepared from commercially available 40 x
2.5 cm. plate glass strips that have been washed with detergent, rinsed in distilled
water and alcohol, and dried with lint-free paper.

● Cleaned strips are clamped into a knife maker, scored with a tungsten carbide wheel,
cracked to form 25 x 25 mm square pieces, and further broken into two triangular
shaped knives using even pressure.

● Glass knives should be prepared and stored in dust-free boxes with lids, just before
use, to avoid contamination.

2. DIAMOND KNIVES

● Diamond knives are used to cut any type of resin block for electron microscopy.

● When supplied by manufacturers, they are already mounted in a metal block

designed to fit directly into the knife holder of the ultrathin microtome.

● Diamond knives are brittle and expensive, but very durable, and the cutting edge
must be kept clean to make it cut longer and to avoid damage during sectioning
 SECTION ADHESIVES

● An adhesive is a substance which can be smeared on to the slides so that the

sections stick well to the slides.

● Most of the tissue sections which are adequately thin and thoroughly dried
without any air bubble trapped under them do not require an adhesive, as in
case of routine H and E staining, but for histochemical methods requiring
alkaline solutions eg ammonia tend to remove sections from slide for such
cases adhesive is required.

● Also adhesive is required for tissues like brain, spinal cord, blood clot,
decalcified tissues which have a tendency to detach themselves from the
slide.

● Tissue impregnated with ester wax also require section adhesive

SECTIONING

● Accomplished by using a cutting apparatus called a microtome.

● Aim: The objective is to produce a continuous “ribbon” of sections adhering to one

another by their leading and trailing edges.

● The thickness of the sections can be preset, and a thickness between 5 - 10 μm is

optimal for viewing with a light microscope

FLOATATION

● Expand the section to its original dimensions and ensure that it is completely flat.

● The temperature will need to be 5 - 9 ˚C below the melting point of the wax. Make
sure the water is clean and free of bubbles.

● To promote efficient drainage and to prevent the section from slipping down the slide,
remove slides vertically from the water

TYPES OF ADHESIVE

➢ Albumin
➢ Gelatin
➢ Starch
➢ Cellulose
➢ Sodium silicate
➢ Resin
➢ Poly L Lysine

➔ Adhesive are either added to water bath or smeared thinly on slide

 TOPIC # 2 HISTOPATH LAB: STAINING

HAEMATOXYLIN AND EOSIN STAINING

● Each procedure is standardized as per requirement
● Work bench aids - used as staining involves multiple steps
●

● Nuclei of Tissue stain - BLUE

● Cytoplasm stain shades - PINK TO RED
 TOPIC # 3 HISTOPATH LAB: MOUNTING

Characteristics Of A Good Mounting Medium:

1. It Should Be Colorless And Transparent.
2. It Should Be Freely Miscible With Xylene And Toluene.
3. It should not dry to a non-stick consistency and harden relatively
quickly.
4. It should protect the section from physical damage and chemical
activity(oxidationandchangesinpH).
5. It Should Be Resistant To Contamination (particularlymicroorganism
growth).
6. It Should Not Cause Shrinkage And Distortion Of Tissues.
7. It should not leach out any stain or affect staining.
8. It should not change color or pH.
9. It Should Be Compatible With The Adhesive In Use