Section Cutting

Microtomy (tissues are placed in a certain wax and become uniform in slices)
- 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.

Microtome – machine/instrument designed for the accurate cutting of thin slices sections of tissues.

Types of Microtome:
a. Sliding microtome– for cutting celloidin (rarely formed serial sections because of its thickness and utilized
for whole eye sections and large section of brain which is tough) embedded sections. (It was named by Adams
(1789).
Two types of microtome:
1. Standard Slide Microtome
2. Base Sledge Sliding Microtome
b. Rotary – for cutting paraffin embedded sections (the most commonly utilized in the lab, most popular, it is
also the one placed inside the cryostat) (Named by Minot [1885 – 1886])
- most popular and most common
c. Rocking/Cambridge – for cutting serial sections of large blocks of paraffin embedded tissues with specific
cutting edge and style (Most basic and now utilized in the lab) (Introduced by Paldwell Trefall – 1881)
d. Freezing- for cutting unembedded frozen sections (not placed in a wax and gelatin with the use of carbon
dioxide supply) (Named by Queckett – 1848)
e. Ultrathin – for electron microscopy work (Named by German people – early 1900’s)
f. Cryostat - For cutting frozen sections (Named by Wilhelm His in 1865 – 1866)

3 Essential parts (same major parts)

1. Block holder - where the tissue is held in position.
2. Knife and knife carrier - for actual cutting of tissue sections as the block is moving towards the knife (block
moves and knife is stationary)
3. Pawl, ratchet feed wheel and micrometer screw - to line up the tissue block in proper position with the knife,
adjusting the proper thickness of the tissue for successive sections (uniform slices)

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

Rocking (Cambridge)

 The most basic/simplest among all of the microtomes.

 It cuts in an arc of circle motion.
 This consists of a heavy base and two arms; the lower arm resting on pivots and a supporting column, and attached
to the micrometer screw, at the base of which is found the ratchet wheel with feed mechanism.
 A section is cut as the tissue passes to the knife edge in a slightly curved plane, in 10 - 12 um thickness.
 The Cambridge rocking microtome, available in two sizes, has been 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 (but if
practically, it can be utilized for serial sections)
 It is not currently favored by most laboratories because of the restrictions in size of tissue block that can be cut, and
the difficulty of reorienting the block.

Rotary (Minot) Microtome

  Most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded
tissues.
 It can be electrically driven. (Leica)
 It is different from the rocking microtome in that the knife and the block holder are brought together by upward
and vertical motions, cutting sections in a perfectly flat plane, thereby allowing excellent serial sections to be
cut.
 It may be used for cutting celloidin blocks of tissues although results are better when the sliding microtome is
used.
 Usually cuts tissue sections in 3 to 5 um.
 The knife is placed in a blade - up position and is therefore relatively dangerous.
 Both manual and electric types can be utilized for cryostat/ultrathin cuts of sections.

Sliding microtome (most dangerous microtome)

 Has 2 types
 Base Sledge Microtime - 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 backwards and forwards under the knife.
 The Base-Sledge microtome is favored in laboratories where very hard tissue or large blocks are usually sectioned.
 Can be used for cutting sections embedded in all forms of media.
 It was originally designed for cutting sections of very large blocks (whole brains).
 Sections are cut in a perfectly flat plane, thereby making excellent serial tissue sections.
 Standard sliding microtome - is different from the base sledge microtome because with this instrument, the block
remains stationary while the knife is moved backward and forward during the process of sectioning.
 It is inherently more dangerous because of the movable knife, which makes it difficult to attach knife guards.
 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.

Freezing Microtome

 The stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe
thru which carbon dioxide passes from a cylinder.
 It is used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues (no application of alcohol),
especially in instances when rapid diagnosis is required, when histological demonstration of fats is needed, when
certain neurological structures are to be studied, and when sensitive tissue constituents to be studied are damaged or
destroyed by heat.

Ultrathin microtome

 An ultrathin microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically
60 to 100 nanometers) of tissue embedded in epoxy resin.
 Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a Transition
electron microscope.
 The ultrathin microtome is also used with its glass knife or an industrial grade diamond knife to cut semi-thin sections
prior to thin sectioning.
 These semi-thin sections are generally 0.5 – 1 μm thick and are mounted on a glass slide and stained to locate areas of
interest under a light microscopy prior to thin sectioning for the TEM.
 Thin sectioning for the TEM is often done with a gem quality diamond knife.

Care of the Microtome

 Accumulated paraffin must be removed from the knife with the use of camel hair brush/ squirrel hair brush
 After drying, wipe the knife and microtome with xylol. Be careful not to wipe the painted parts because the
xylol will remove the painted parts.
 To prevent rusting, movable portions must be oiled.
 To avoid accumulation of dust, cover the microtome.
 The microtome must be away from airdrafts, hallways/corridors, moving staff.
 Always remove the knife or blade before cleaning (may damage the cutting edge of the knife)
 When cleaning the blade avoid dragging anything along the cutting edge.
 Even cellulose fibers can cause damage to the blade.
 Have the instrument inspected at least once a year by a qualified service technician from the manufacturer

Serial sections
– unbroken sequence of sections throughout the tissue blocks.
- all sections are saved.

Freezing microtomes have been replaced in most laboratories by the cryostat, which is easier to operate (optimum
temp = -18 to – 20 degrees centigrade = cold microtome procedure)
╚> faster, and gives thinner sections.

Frozen sections may be cut from fixed or fresh tissue, but generally fixed enzyme preparation.
Microtome Knives:
1. Plane concave
Either:
- less concave (straight) for celloidin
- concave for paraffin sections
***applicable to rocky, rotary, standard sliding microtome and base sledge microtome
– 25mm in length
- one side of the knife is straight while the other is concave

2. Biconcave
– 120mm in length
- with both sides concave (for paraffin = rocking and rotary microtome)

3. Plane wedge (for celloidin sections = standard sliding microtome and base sledge microtome)
– 100 mm in length
- have both sides straight
Today, most knives used in section cutting are wedge – shaped (theoretical) but know, in practice we are using
razor blades. The sides of the wedge knives are inclined at an angle of approximately 150 and the surfaces of
these are highly polished so that sections will not adhere to them but will move on the surface, thus minimizing
folding, distortion, and sticking and facilitating good ribbon formation.

 The actual cutting part of the knife forms an angle which is known as the “bevel angle”, and is normally about 27-
32 0.
***Clearance angle = 5 – 10 degrees
 The angle of these facets is kept constant for each knife by the provision of a slide-on back for use on each knife
during the processes of honing and stropping.

Sharpening of Microtome Knives

* The degree of sharpness is proportional to the fineness of the abrasive used in sharpening.
 * The final sharpening materials used must have a grain size smaller than the permissible serrations remaining in
the edge.

* Sharpening may be carried out either by hand or machine.

HARD SHARPENING – done in 2 ways:

a. By hones
b. By glass plates (utilized for ultrathin – EM) and abrasives

Hones – a natural stone or a hard grinding surface for sharpening a knife or other cutting tools.
* Good quality hones are expensive, but only the best quality should be used.
* The finer the grain in a hone, the harder the hone.
1. Carborundum – available in a wide variety of fineness
2. Arkansas hone – a stone of medium fineness
3. Yellow Belgian -the finest
4. Belgian Black vein (blue green)
- also called oil stones  because oil is commonly used as a lubricant

Many oils may be used:

a. light machine oil
b. Liquid paraffin oil (mineral oil)
c. Vegetable oil
d. Xylol – tends to evaporate too rapidly
e. Natural oil – become messy

Additional hones

 A flat circular glass plate with finely powdered aluminum oxide made into paste with water (used as an abrasive)
may be used for grinding and removing nicks.

 Diamantine may also be used for final polishing. The plate glass is usually ¼ - 3/8-inch-thick, about 14 inches
long and 1-2 inches wider than the length of the knife blade to be sharpened.
Honing – involves the removal of gross nicks on the knife (coarse honing) to remove blemishes, and grinding the
cutting edge of the knife on a stone to acquire an even edge.
* Direction: Heel to toe
* Purpose: To remove irregularities and nicks on the knifes edge

Stropping – the process whereby the “burr” formed during honing is removed and cutting edge of the knife is polished.
* Direction: Toe to heel
* Purpose: To sharpen and polish the knife

Heel to Toe

 The knife is fitted to its corresponding back, placed on one end of the hone, and with the cutting knife edge first, the
"heel“ (handle end) is drawn obliquely or diagonally towards the operator on the stone until the "toe" (head portion)
is reached.
 The knife is then turned over, and the other surface is again drawn forward, EDGE FIRST, with a HEEL TO TOE
direction.

Toe to Heel

 The procedure is the reverse of honing.

 The knife is first fitted with its appropriate knife back, then laid obliquely on the strop and with the cutting edge
behind, (EDGE LAST) is pushed backward and drawn forward in a TOE TO HEEL direction.
 Around 40-120 double strokes are usually required.
 Types of Tissue Sections:
1. Paraffin sections
 for paraffin embedded tissue blocks
 may be cut by rotary and rocking
 4 - 6 µ thick

2. Celloidin sections
 for celloidin embedded tissues
 usually made by means of the sliding microtome
 10 - 15 µ thick

3. Frozen sections – may be cut from tissues that have been fixed and frozen with CO 2 (Cold Knife procedure) or
for fresh or fixed tissues frozen with the cryostat (cold microtome)

Other 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.

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.
 These 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.

Float – out/ water bath (Floating out of sections = will make the tissue visible and flatten the tissue)
- convenient to have such a bath close by the microtome.
- a circular, thermostatically controlled bath 10-12 inches in diameter and 3-4 inches in depth is desirable
- the inside surface should be black, thus enabling easier visualization of sections.
- the thermostat should be set at 45 0C, i.e. about 10 0 below the melting point of the wax used for blocking out.

Forceps (fine pointed or curved) and squirrel hair brush

-These tools are needed for handling sections during cutting, and for removing folds and creases on the sections during
"floating out" in water bath.
PARAFFIN SECTION

Drying Sections on Slides

- after draining, the sections must be fixed to the slides. This may be done in several ways:

a. In a wax oven at 56 – 60 0C for 2 hours

b. In an incubator at 37 0C overnight
c. On a hotplate at 45-55 0C for 30-45 minutes preferably inverted, the slide ends resting on tracklike support 3-
4mm above the plate surface.
d. For 20 – 30 minutes at 50-55 0C in a slide dryer (blow type)

PARAFFIN SECTION

Drying Sections on Slides

  By mounting the sections in 0.1 -0.25% aqueous floating out gelatin solution of or onto albuminized slides,
draining excess fluid, and while still moist, placing them in a covered coplin jar containing 2-3 mL 40%
formalin in the incubator at 37 0C for 4-18 hours.

 In urgency, by carefully holding the slide section upward above a Bunsen burner until wax just melts.

FROZEN SECTIONING
Advantages of Frozen and Cryostat Sections
1. For certain staining procedures:
e.g. - demonstration of fat by the Oil Red O method
- Sliver impregnation method
- For certain methods in the CNS (Central Nervous System)

2. Frozen or cryostat sections are indispensable for rapid diagnosis during operations.

3. All enzymes are destroyed at temperature above 560C and although some (phosphatases) may be demonstrated
in paraffin sections, all are best shown in cryostat or frozen sections of fresh tissues.

Cryostat
- advantage over freezing microtomes: it maintains the tissue block, knife and section at the same temperature
- optimum working temperature: -18 to -200C
- cuts individual sections  does not ribbon
- tissues will also adhere easily to the glass slide without need for any adhesive mixture.

* Fixed tissue may be used.

 the tissue block is selected by the pathologist
 may be immersed in boiling 10 % BNF for ½ -2 minutes before freezing and sectioning for rapid surgical
diagnosis.

Staining of Cryostat Sections

for rapid surgical diagnosis, 2 methods are widely used:
a. H&E (Hematoxylin and Eosin
b. Polychrome methylene blue