LESSON 12: TRIMMING AND MICROTOMY

 TRIMMING
 The process of cutting the excess to expose the tissue surface in preparation for actual microtomy.
 Sides, to and bottom are trimmed until perfectly level and all sides parallel
 Knife/blade may be used

 MICROTOMY
 The process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into
uniformly thin slices or “sections” to facilitate studies under the microscope. It is known as microtomy.
 The cutting of processed tissue into uniformly thin slices or sections and attched to the slides for further
microscopic examination.
 The basic instrument used is a microtome that is capable of cutting a section at a predertemined thickness by
sliding the block into a cutting tool, usually a steel knife of blade, which is fixed and attached to the machine.

THREE ESSENTIAL PARTS OF MICROTOME

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 adjustments screw
 To line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for
successive sections

Kinds of Microtome
1. Rocking microtome
USE: For cutting serial sections of large blocks of paraffin embedded tissues
AKA. Cambridge rocking microtome
 INVENTED BY: Paldwell Trefall (1881)
 Simplest among different types of microtomes
 Thickness of tissue ribbon produced: 4um
 Disadvantage: block is difficult to reorient

2. Rotary microtome
USE: for cutting paraffin embedded sections
 INVENTED BY: Minot (1185-1886)
 Most common type of used in routine and research laboratories
 Most common type housed in cryostat
 Thickness of tissue ribbon produced : 4-6um
 Advantage: ability to cut 2-3mm sections

3. Sliding microtome
USE: for cutting celloidin embedded
 INVENTED BY: Adams (1879)
 Most dangerous type of microtome. (state the reason)
2 types of Sliding Microtome
A. Base Sledge - knife is stationary; tissue block is moving
 Consist of two movable pillars holding the adjustable knife clamps, allowing the knife to
be set at an angle for cutting celloidin sections
 Suited for sectioning specimens embedded in all forms of media, especially for cutting
sections from tough tissue blocks which may offer great resistance to the knife

B. Standard Sliding- knife is moving; tissue block is stationary

 is developed mainly for cutting celloidin embedded tissue blocks and inherently more
dangerous because of the movable knife, which makes it difficult to attached knife guards
  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
USE: for cutting unembedded frozen Sections
 INVENTED BY: Queckett (1848)
 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 damaged or destroyed by heat.

The Cryostat or Cold Microtome

 CRYOSTAT is a refrigerated apparatus used in fresh tissue microtomy, for freezing the tissue into the block
holder to the correct degree of hardness to facilitate easier and faster sectioning.
It consist of :
 Microtome -usually a rotary microtome; ept inside a cold chamber which have been maintained at a
temperture between -5C to -30 C (average is -20C) by an adjustable thermostat, capable of freezing
fresh tissues within 2-3 minutes and cutting sections of 4 u with ease.
 It provides a means of preparing thin sections of frozen tissues especially for fluorescent antibody staining
techniques and histochemical enzyme studies.
 Sections are usually transferred directly from a microtome knife to alide or coverglass, all of which are
maintained at a low temperature.

5. Ultrathin microtome
USE: for cutting sections for electron microscopy
 Fixative: osmium tetroxide
 Embedding medium: Epoxy resins
 Thickness of tissue ribbons produced: 0.5um
 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 glass or
diamond knives
 The specimen used is small, fixed in osmium tetroxide, and embedded in plastic

MICROTOME KNIVES
Trimming and section cutting are done with microtome knife, which is available in three basic types or shapes:
1. Plane- concave knife
 - usually 25mm in length
 - one side of the knife is flat while the other is concave
 Less concave- celloidin embedded tissue on a sliding microtome
 More concave- paraffin embedded tissue using rotary and rocking microtome.

2. Biconcave knife
 - usually 120mm in length
 - both side are concave
 -recommended for cutting paraffin embedded sections on a rotary microtome

3. Plane wedge knife

 - usually 100mm in length
 - both sides are flat/straight
 - recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin
blocks, using a base sledge type sliding microtome.

Remember!
BEVEL ANGLE-angle formed between the cutting edge = 27-32degree
CLEARANCE ANGLE- angle formed between the cutting facet presenting to the block and the surface of the
block= 0-15 degree
WEDGE ANGLE- angle formed by the sides of the wedge of knife= 14 degrees

Care of 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 microtome, since this may later on interfere with the cutting of tissue block
 After careful drying the machine and knife holder, the parts should be wiped with xylol.
 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 the tissues.

HONING AND STROPPING

 Badly nicked knives with blunted ends have to undergo sharpening in order to ensure optimum sectioning of
tissue blocks and prevent gross irregularities on the tissue sections.

Sharpening of Knife involves Two Stages:

1. 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
- the degree of sharpness is proportional to the fineness of the abrasive used in sharpening.
PURPOSE: remove irregularities from the knife
Direction: Heel to Toe
Hones- a natural sharpening stone or hard grinding surface

Several Examples of Hones

A. Belgium Yellow
 for manual sharpening when cutting edge has been rendered blunt or nicked.
 Gives the best result
B. Arkansas
 gives more polishing effect than the belgium yellow
C. Fine Carborundum
- much coarser than the two types used only for badly nicked knives followed by either one of the first two
knife sharpeners.
D. Plate-glass hone
E. Machine Hone

COMMON LUBRICANT USED FOR HONING

1. Mineral oil
2. Clove oil
3. Xylene
4. Liquid paraffin
5. Soapy water

NOTES TO REMEMBER:
 Edge first: heel to toe direction, 10 – 20 strokes.
 Mechanical honing make use of a vibrating frosted glass plate or wheel driven by an electrical motor
PRECAUTIONS:
 The hone should be long enough (8” x 3”) to allow the whole length of the knife edge to be sharpened in a
single stroke and wide enough to sufficiently support and prevent the rocking of the knife.
 The hone should be lubricated with soap water or fine oil before using. It is then washed, preferably with
water, to remove all metal particles that may have been collected during the process.
 The hone should be cleaned before, during and after use.
2. STROPPING
 the process whereby the “burr” formed during honing is removed and the cutting edge of the knife is polished
PURPOSE: to polish and sharpen the cutting edge
 if the knife has become dull and blunt, but is free from nicks or teeth, it is usually only necessary to strop it.
 -the process of stropping:(edge last) toe to heel, 40 -120 double strokes
 Fine quality horse leather is used as paddle strops which may be either flexible or rigid.

PRECAUTIONS:
 Knife should always be wiped clean with a soft cloth before and after a series of stropping strokes and before
changing from a coarse to a fine strop to remove particles which may have been taken off the knife.
 Speed in stropping should be avoided.
 Mineral oil is not recommended and should never come in contact with a strop since it will tend to blister and
destroy the leather
 Stropping surfaces should be firm and not loose, to prevent the turning of the knife’s edge
 Wax must not be allowed to come in contact with the strop
 Never wipe with paper or cloth on the knife

DISPOSABLE BLADES
 Due to the availability of disposable blades nowadays, honning and stropping are no longer common practice
in the laboratories.

GLASS KNIVES
 Are generally used for trimming and semi-thin sectioning of tissue blocks for electron microscopy.

DIAMOND KNIVES
 Are used to cut any type of resin block for electron microscopy.

OTHER EQUIPMENT
 In addition to microtome and microtome knife, the following are also required during the process of sectioning.

1. WATERBATH
 The temperature of water should be about 10C below the melting point of the paraffin wax.
 A small amount of detergent may be added to the water to reduce the surface tension and allow section to
flatten out.

2. DRYING OVEN OR HOT PLATE

 Small drying ovens are now available with temperature setting at the melting point of wax to allow
sections to dry.
 A hot plate may be also used instead of drying oven
 For more delicate tissues such as brain, a lower drying temperature is used to avoid the splitting and
craking of section due to excessive heat.

3. FORCEPS (fine pounted or curved) and SQUIRREL HAIR BRUSH

 Used for handling sections during cutting
 Used for removing folds and creases on the section during “floating out” in the water bath.

4. CLEAN SLIDES
 76x 25mm slides that are 1.0-1.2mm thick are preffered beacuse they do not break easily.
5. ICE TRAY

6. PENCIL