PREFINALS: TRIMMING AND MICROTOMY Flashcards
Removal of excess wax using knife or cutter after the wax block is removed from the tissue cassette or paper boat
TRIMMING
Formation of truncated pyramid and exposure of the tissue surface for ease of sectioning
TRIMMING
Allow tissue blocks to fit into the block holder of microtome
TRIMMING
At least 2mm should surround the tissue block
TRIMMING
TRIMMING 2 TYPES
o Coarse Trimming
o Fine Trimming
AKA Sectioning
MICROTOMY
Formation of uniformly thin slices/sections/ribbons from the tissue block with the use of a microtome in order to facilitate studies under the microscope
MICROTOMY
Sections usually form ribbons due to slight heat generated between the block and the knife edge during the process of cutting
MICROTOMY
Complete ribbons are picked up with camel hair brush, forceps or fingers.
MICROTOMY
Principle: Spring-balanced 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.
Microtome
Microtome Basic Parts:
Block holder/chuck/cassette clamp
Knife Carrier and Knife
Pawl, Ratchet Feed Wheel and Adjustment Screws
where the tissue is held in
position
Block holder/chuck/cassette clamp
for actual cutting of tissue sections
Knife Carrier and Knife
to line up the tissue block in proper position with the knife, and to adjust the proper thickness of the tissue
Pawl, Ratchet Feed Wheel and Adjustment Screws
Brush away accumulated paraffin and small pieces of tissues with _______ after sectioning
soft brush
Excess paraffin and tissues may later on ______ with the cutting of tissue blocks
interfere
may also be used for cleaning some parts of the microtome
Xylene
_____ to prevent rusting.
Oil movable parts
______ to prevent accumulation of dust.
Cover microtome
The cutting rate depend upon the type of tissue, the size of the block and the model of the microtome that is used
TRUE
Rocking or Cambridge
Inventor:
Paldwell Trefall
Simplest
Rocking or Cambridge
Rocking or Cambridge
Cutting section:
10-12µm
For small and large paraffin blocks
Rocking or Cambridge
not for serial sections because of slightly curved planes
Rocking or Cambridge
Rotary Inventor:
Minot
Most common
Rotary
Rotary Media:
Paraffin
Excellent for serial sections
Rotary
Sliding Inventor:
Adams
Sliding Media:
Celloidin
Very hard and rough tissue blocks
Sliding
Sliding Types:
o Base-sledge
o Standard sliding
more dangerous d/t moving knife
Standard sliding
Freezing Inventor:
Queckett
Frozen sections (undehydrated tissues like fat) for rapid diagnosis
Freezing
Stage for block holder is hollow and perforated, attached to a flexible lead pipe containing carbon dioxide.
Freezing
CO2 as propellant/freezing agent (2 to 3 minutes)
Freezing
Cryostat/Cold Microtome
More common than freezing microtome
Cryostat/Cold Microtome
STAT frozen section (intraoperative diagnosis)
Cryostat/Cold Microtome
Chamber maintained -5 to -30OC with rotary microtome inside
Cryostat/Cold Microtome
Cryostat/Cold Microtome Cutting Section:
4 µm
Microtome Knives:
- Plane-Concave
- Biconcave
- Plane-Wedge
Length
Plane-Concave
25mm
Length
Biconcave
120mm
Length
Plane-Wedge
100mm
Characteristic
Plane-Concave
- One side is flat
- Other side is concave
Characteristic
Biconcave
Both sides concave
Characteristic
Plane-Wedge
Both sides straight
Embedding Medium
Plane-Concave
- Celloidin
- Paraffin
Embedding Medium
Plane-Concave:
Less concave
Celloidin
Embedding Medium
Plane-Concave:
More concave
Paraffin
Embedding Medium
Biconcave
Paraffin
Embedding Medium
Plane-Wedge
Paraffin
Microtome
Plane-Concave
- Sliding
- Base-sledge, Rotary or Rocking
Microtome
Plane-Concave:
Celloidin
Sliding
Microtome
Plane-Concave:
Paraffin
Base-sledge, Rotary or Rocking
Microtome
Biconcave
Rotary
Microtome
Plane-Wedge
Base-sledge
A good cutting edge must be able to cut good sections from a paraffin wax block about ____, without any serration
2-3microns thick
likely to produce nicks or jagged edges
Too hard cutting edges
likely to become easily dull
Too soft cutting edges
Angles:
- Bevel Angle: 27° to 32°
- Cutting angle (15°)
- Clearing Angle
Angle between the cutting edges
Bevel Angle: 27° to 32°
Maintained by slide-on back (spring-loaded, semi-circular metal sheet slipped onto the knife)
Bevel Angle: 27° to 32°
angle between the face of a cutting tool and the surface of the work
Cutting angle (15°)
- knife should be inclined at 5-10° from the cutting plane so that the cutting facets (bevel angle) will not compress the block during the process of cutting.
Clearing Angle
Other Knives and Blades:
- Disposable Blades
- Glass Knives/Ralph Knives
- Diamond knives
- Safety razor blades
Widely used now because cheaper; honing and stropping are no longer common practice;
Disposable Blades
coated with polytetrafluoroethylene (for ease of ribboning)
Disposable Blades
E.g. Magnetic knives in cryostat
Disposable Blades
For ultrathin microtomes
Glass Knives/Ralph Knives
For resin blocks on ultrathin microtomes; brittle and expensive
Diamond knives
Easily replaced when dull and produces good tissue sections same as with microtome knives
Safety razor blades
For partially calcified materials, paraffin and frozen sections.
Safety razor blades
Unsatisfactory for sections less than 10micra
Safety razor blades
Badly nicked knives with blunted ends have to undergo sharpening in order to ensure optimum sectioning of tissue blocks
SHARPENING
Sharpening of the knives involves 2 stages, namely:
1) Honing
2) Stropping
Removal of nicks and irregularities on the knife edges
Honing
Natural sharpening stone or hard grinding surface
Hones
Wide enough to sufficiently support and prevent the rocking of the knife
Hones
Long enough to allow the whole length of knife edge to be sharpened in a single stroke
Hones
TYPES OF Hones:
- Belgium Yellow
- Arkansas
- Fine Carborundum
- Plate Glass (8x3x1in)
most common; best result
Belgium Yellow
has more polishing effect
Arkansas
coarser; for badly nicked knives
Fine Carborundum
excellent
Plate Glass (8x3x1in)
Lubricants
o Soapy water
o Mineral oil
o Clove oil
o Xylene
o Liquid paraffin
May be used for grinding and removing nicks
Flat Glass plate with finely powdered aluminum oxide
For final polishing
Diamantine
Procedure: Heel to Toe movement, Edge first
Honing
Procedure: Heel to Toe movement, Edge first:
- Clean hone with xylene to remove scattered particles of stones and metal
- Cover with lubricant
- Knife is fitted to its corresponding back, placed on one end of the hone with cutting edge first
- With 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.
- Honing is then continued until all the teeth in the knife edge have been eradicated”
- Washed with water after using to simply remove the metal collected during the process
- After honing, wipe off the oil or soap from the knife with xylene, then strop it thoroughly……
_____ per surface for Minot or Plane-wedge knife
10-20 strokes
For plane-concave knives,_____
only the concave surface should be rubbed on the hone
Plane-wedge & plane-concave is provided with “backs” to maintain the ____ throughout honing
correct bevel angle (27-32°)
Utilizes a machine that make use of vibrating frosted slide plate or wheel driven by electrical motor.
Mechanical Honing
The knife is pressed against the flat side of a rotating glass wheel.
Mechanical Honing
30 double strokes - given to each side of the knife.
Mechanical Honing
Advantages: Time-saving; produce well sharpened knives with uniform bevels
Mechanical Honing
Disadvantage: Expensive
Mechanical Honing
Removal of burrs and polishing of cutting edge
Stropping
Stropping Materials:
Paddle strop made of horse leather attached to a solid back, in order to prevent sagging
Usually dry thus require oiling
Paddle strop made of horse leather attached to a solid back, in order to prevent sagging
Vegetable oil (e.g. castor oil) applied on the back of the horse leather
Paddle strop made of horse leather attached to a solid back, in order to prevent sagging
Not mineral oil because it tends to blister and destroy the leather
Paddle strop made of horse leather attached to a solid back, in order to prevent sagging
Procedure: Toe to Heel movement, Edge Las
Stropping
The procedure is the reverse of honing
Toe to Heel movement, Edge Las
Procedure: Toe to Heel movement, Edge Last:
- The knife is fitted with its appropriate knife back
- Knife is laid obliquely on the strop and with the cutting edge behind
- Edge last is pushed backward and drawn forward
Procedure: Toe to Heel movement, Edge Last
Precautions:
The knife should always be wiped clean with a “soft” cloth before and after series of stropping (NEVER use paper or cloth)
The knife edge is the oiled or greased to preventing it from rusting
Pressure during the first stropping strokes should be quite light since the natural compressibility of the leather is what actually does the work
Speed in stropping should be avoided
Wax must not be allowed to come in contact with the strop.
40-120 double strokes:
- Plane wedge knife
- Plane – Concave knife
both sides are required for stropping
Plane wedge knife
only the concave surface should be stropped
Plane – Concave knife
TYPES OF TISSUE SECTIONS:
- Paraffin
- Celloidin
- Frozen Sections
4-6µm
Paraffin
Successive sections will usually stick edge-to-edge (knife) due to local pressure with each cutting stroke, thereby forming a ribbon. (remedy: cut slowly)
Paraffin
Sections are removed in ribbons of ten to allow easy location of serial sections.
Paraffin
The blocks are trimmed in the same manner as in paraffin blocks
Celloidin
To avoid dehydration and shrinkage, sections are usually cut by the wet method, both the sections and the block being kept moist with 70% alcohol during cutting.
Celloidin
Celloidin sections do not come off in ribbons and have to be collected into 70% alcohol immediately
Celloidin
Methods of preparing frozen section
Frozen Sections
Frozen Sections types:
1) Cold knife procedure
2) Cryostat procedure (cold microtome)
10-15µm
Celloidin
Sections are floated out on a water bath set at 45-50°(approx. 6-10°C lower than the melting point of the wax used for embedding the tissue.)
FLOTATION/FLOATING-OUT
For paraffin sections
FLOTATION/FLOATING-OUT
5 to 10OC↓MP of Wax
Flotation Bath
Inside is specifically colored enamel black
Flotation Bath
TSEs flatted after 30sec; removes tse wrinkling
Flotation Bath
Flotation Bath
Dimensions:
d=11in, h=4in, 2L capacity
Slides SIZE:
76x25mm, 1-1.2mm thick, frosted)
Regulated temp. to flatten the sections and prepare them for mounting into the slides/slider
T
Sections should not be left on the water bath for a long time (30 seconds will be enough) to avoid undue expansion and distortion of the tissue
T
___may be used for adhesion of tissue to the slide
ADHESIVES
may be removed by stretching the
sections gently
Folds or creases sections
may be teased out beneath the sections by means of needle
Bubbles
Selected sections for staining should be fished out in a vertical position
FLOTATION/FLOATING-OUT
Mounted sections are placed in a paraffin oven to dry
DRYING THE SLIDES
45 – 55°c for:
- enzyme digestion
- chemical extraction
- metallic impregnation
- enzyme localization technique
Hot plates are not recommended because they can cause:
Overheating
Dust falling – onto the section during drying period
Metal racks with 25-slide divisions are used to store the mounted sections during the drying process which usually takes 5 minutes in the heated oven. Once dry, the whole rack of slides can be taken for manual staining.
DRYING THE SLIDES
