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Trimming and Sectioning

The document discusses trimming and sectioning of tissue samples using a microtome. It describes how a microtome works by using a knife to cut very thin uniform sections from a tissue block. There are different types of microtomes for cutting various samples, including rocking, rotary, sliding, freezing, and ultrathin microtomes. Good technique and knife maintenance are important to obtain high quality sections. Sections are mounted on slides and may require treatments before staining.

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Pia Pascual
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312 views22 pages

Trimming and Sectioning

The document discusses trimming and sectioning of tissue samples using a microtome. It describes how a microtome works by using a knife to cut very thin uniform sections from a tissue block. There are different types of microtomes for cutting various samples, including rocking, rotary, sliding, freezing, and ultrathin microtomes. Good technique and knife maintenance are important to obtain high quality sections. Sections are mounted on slides and may require treatments before staining.

Uploaded by

Pia Pascual
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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TRIMMING and SECTIONING

TRIMMING
• Process of removing excess wax from the block to
expose the tissue surface in preparation for actual
cutting
• Sides, top and bottom of tissue block are trimmed
SECTIONING
• Process by which a processed tissue is cut into
uniformly thin slices (sections) with the aid of a
machine called Microtome
• Sections are cut between 4-6u in thickness
THREE ESSENTIAL PARTS:
1. Block Holder
2. Knife Carrier and Knife
3. Pawl, Ratchet Feed Wheel and 3.

Adjustment Screws 1.

2.
PRINCIPLE OF MICROTOME
• Spring-balanced teeth or pawl 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.
FIVE KINDS OF MICROTOMES
1. Rocking microtome – Serial section of large blocks
2. Rotary microtome – Paraffin embedded
3. Sliding microtome – Celloidin embedded
4. Freezing microtome – Frozen sections
5. Ultrathin microtome – Electron microscope
ROCKING MICROTOME
• Invented by: Paldwell Trefall in
1881
• Simplest
• Used to cut small and large block
of paraffin
• Disadvantage: difficulty in
reorienting the block
• Section is cut 10-12 u thickness
ROTARY MICROTOME
• Invented by: Minot in 1885
• Most common
• Cut paraffin embedded
• Ideal for serial sections
• Section is cut 3-5 um thickness
SLIDING MICROTOME
• Invented by: Adams in 1789
• Most dangerous
• Two types:
– Base – sledge microtome
• Block holder: moving
• Knife 24cm long: stationary
– Standard Sliding microtome
• Block: Stationary
• Knife: moving
• Celloidin sections
FREEZING MICROTOME
• Invented by: Queckett in 1848
• Uses to cut undehydrated
tissue in frozen state
• Cryostat or Cold Microtome
– Refrigerated apparatus
– Freezing the tissue to the correct
degree of hardness
– Rotary microtome
– Temp: -5° to -30°C (-20°C)
– Section is cut 4 u
ULTRATHIN MICROTOME
• Invented by: Marfred Von Ardenne
• cutting sections for electron
microscopy
• Diamond Knives - cut any type of
resin block, mounted in a metal
block
• Glass Knives - trimming and semi-
thin sectioning of tissue blocks
• Section is cut 0.5 to 1 um thick
CARE OF THE MICROTOME
• Soft brush - used to brush away small pieces of tissue
• Parts should be wiped with Xylol
• Prolonged xylene - capable of removing paint
• Moving parts: Oiled - to prevent rusting
Microtome Usual Description
Knives Length
Plane Concave 25 mm One side: Flat
Other side: Concave

Celloidin embedded – Sliding microtome


Paraffin embedded – Rotary/Rocking microtome

Biconcave 120 mm Both sides: Concave

Paraffin embedded – Rotary microtome

Plane wedge 100 mm Both sides: Straight

Frozen sections or hard spx in paraffin blocks –


Base-sledge microtome
CHARACTERISTICS OF GOOD CUTTING EDGE
• Good quality steel
• Too soft: dull easily
• Too hard: nicks/ jagged edges and irregularities
• Must be able to cut good sections about 2-3 microns thick
• Must be sharp enough to cut paraffin wax block at 4 μ thick without
serrations

CUTTING FACET: “BEVEL”


BEVEL ANGLE –27°to 32°
CLEARANCE ANGLE
– 15° - Perfect and optimum cutting angle
– 5° to 10° - To prevent uneven sections
HONING – HEEL TO TOE
(SHARPENING)
• Coarse Honing – gross nicks
• Honing Proper – even edge
acquired
• Hone – carborundum – 8”x3”
– Belgium Yellow – best result
– Arkansas – more polishing effect
– Fine Carborundum – much coarser
– for badly nicked
COMMON LUBRICANT USED FOR HONING
1. Mineral oil
2. Clove oil
3. Xylene
4. Liquid paraffin
5. Soapy water
STROPPING – TOE TO HEEL
(POLISHING)
• Burrs formed during honing is
removed
• Cutting edge of knife is polished.
• Paddlestrop –made of horse
leather
• 40-120 double stokes
• Treated with vegetable oil/Castor
oil
DISPOSABLE BLADES
• Sharp cutting edge can cut 2-4 u thick
OTHER EQUIPMENT
• Waterbath – 5°-10°C below melting point
• Drying oven or hot plate
• Forceps and brush
• Clean Slides – 76x25mm, 1.0-1.2mm
thick
• Slide rack
CHARACTERISTIC OF A FISHING-OUT
RIBBON
• After cutting, tissue
• Thin sections are floated
• Transparent out on a water bath
• Not wrinkled • When the sections are
• Without irregularities flattened out, slide is
• Uniform in thickness immersed in water
• Can easily be separated bath and tissue is
from each other fished out
• Continuous
METHODS TO FLATTEN
RIBBON
• 20% Alcohol
• Float out bath – 45-
50°C
• Distilled water in
hotplate
ADHESIVES
• Mayer’s Egg Albumin
– Egg White
– Glycerin - to prevent drying
– Thymol - to prevent growth of molds
• Dried Albumin
• 1% Gelatin – mix with water in float out bath
• Gelatin-Formaldehyde
• Starch Paste
• Plasma (0.5mL)
• Poly-L-Lysine - IHC
• APES (3-aminopropylthriethoxysilane) - cytology
DEPARAFFINIZATION
• Paraffin surrounding the tissue section is
removed to prevent over staining of tissue.
METHODS
• Alcohol lamp
• Xylene bath
• Hot oven – 55-60°C
PROBLEM REASON REMEDY

Sections fail to form ribbons Surfaces and edges Re-trim the block
of the block are not
Parallel
Ribbon is curved, crooked or uneven Blunt or dull spot on Adjust the knife so that knife edge will present a uniformly sharp
instead of straight the knife, producing edge to the block, or sharpen
an irregular knife
edge
A hole is formed in the section Bubble or dirt formed Re-embed in freshly filtered wax if Necessary
in the embedding
medium
Sections adhere to the knife or other Static electricity due Breathe out or blow gently on the bock and knife to break up
parts of the microtome to low atmospheric static electricity, or boil water in the room to increase Humidity
Humidity
Horizontal or parallel lines or furrows Knife edge vibrates Treat with phenol during processing or Collodionize
across the section ("chatters") are seen due to hardness of
Tissue
Section cut is sometimes thin, Knife is not clamped Adjust the knife so that knife edge will present a uniformly sharp
sometimes thick properly edge to the block, or sharpen

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