DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: HPCT

TRIMMING
 Removal of excess wax using knife or cutter after the wax block Types of Microtome
is removed from the tissue cassette or paper boat The cutting rate depend upon the type of tissue, the size of the block
 Formation of truncated pyramid and exposure of the tissue and the model of the microtome that is used.
surface for ease of sectioning
 Allow tissue blocks to fit into the block holder of microtome 1. Rocking or Cambridge  Simplest
 At least 2mm should surround the tissue block Inventor: Paldwell Trefall  Cutting section: 10-12µm
o Coarse Trimming  For small and large paraffin
o Fine Trimming blocks
 not for serial sections
MICROTOMY because of slightly curved
 AKA Sectioning planes
 Formation of uniformly thin slices/sections/ribbons from the 2. Rotary  Most common
tissue block with the use of a microtome in order to facilitate Inventor: Minot  Media: Paraffin
studies under the microscope  Excellent for serial sections
 Sections usually form ribbons due to slight heat generated 3. Sliding  Media: Celloidin
between the block and the knife edge during the process of Inventor: Adams  Very hard and rough tissue
cutting. blocks
 Complete ribbons are picked up with camel hair brush, forceps  Types:
or fingers. o Base-sledge
o Standard sliding – more
Microtome dangerous d/t moving
Principle: Spring-balanced or pawl is brought into contact with, and knife
turns a ratchet feed wheel connected to a micrometer screw, which is 4. Freezing  Frozen sections
in turn rotated, moving the tissue block at a predetermined distance Inventor: Queckett (undehydrated tissues like
towards the knife for cutting sections at uniform thickness. fat) for rapid diagnosis
 Stage for block holder is
hollow and perforated,
Microtome Basic Parts attached to a flexible lead
 Block holder/chuck/cassette clamp – where the tissue is held in pipe containing carbon
position dioxide.
 Knife Carrier and Knife – for actual cutting of tissue sections  CO2 as propellant/freezing
 Pawl, Ratchet Feed Wheel and Adjustment Screws – to line up agent (2 to 3 minutes)
the tissue block in proper position with the knife, and to adjust
the proper thickness of the tissue 5. Cryostat/Cold  More common than freezing
Microtome microtome
 STAT frozen section
Care of the Microtome (intraoperative diagnosis)
 Brush away accumulated paraffin and small pieces of  Chamber maintained -5 to -
tissues with soft brush after sectioning 30OC with rotary microtome
 Excess paraffin and tissues may later on interfere inside
with the cutting of tissue blocks  Cutting Section: 4 µm
 Xylene – may also be used for cleaning some
parts of the microtome
 Oil movable parts to prevent rusting. 6. Ultrathin  Cutting section: 0.5µm
 Cover microtome to prevent accumulation of dust.  Media: Plastic
 For electron microscopy,
tissues fixed with osmic acid
 Uses special diamond knife
(ADV: very sharp and no
easy dulling)

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 Microtome Knives SHARPENING
Plane-Concave Biconcave Plane-  Badly nicked knives with blunted ends have to undergo sharpening
Wedge in order to ensure optimum sectioning of tissue blocks.
Length Length: 25mm 120mm 100mm  Sharpening of the knives involves 2 stages, namely:
1) Honing
Characteristic One side is flat Both sides Both 2) Stropping
Other side is concave sides
concave straight
Honing
Embedding Less More Paraffin Paraffin
Removal of nicks and irregularities on the knife edges
Medium concave concave
Materials:
Celloidin Paraffin
 Hones
- Natural sharpening stone or hard grinding surface
Microtome Sliding Base- Rotary Base-
- Long enough to allow the whole length of knife edge to be
sledge, sledge
sharpened in a single stroke
Rotary
- Wide enough to sufficiently support and prevent the
or
rocking of the knife.
Rocking
o Belgium Yellow – most common; best result
NOTE:
o Arkansas – has more polishing effect
 A good cutting edge must be able to cut good sections from a
o Fine Carborundum – coarser; for badly nicked knives
paraffin wax block about 2-3microns thick, without any serration
o Plate Glass (8x3x1in) – excellent
 Too soft cutting edges - likely to become easily dull
 Lubricants
 Too hard cutting edges – likely to produce nicks or jagged edges
o Soapy water
Angles o Mineral oil
o Clove oil
1. Bevel Angle: 27° to 32°
o Xylene
 Angle between the cutting edges
o Liquid paraffin
 Maintained by slide-on back (spring-loaded, semi-circular
metal sheet slipped onto the knife)  Knife sharpeners
1. Cutting angle (15°) - angle between the face of a cutting tool and  Flat Glass plate with finely powdered aluminum oxide
the surface of the work  May be used for grinding and removing nicks
2. Clearing Angle - knife should be inclined at 5-10° from the cutting  Diamantine
plane so that the cutting facets (bevel angle) will not compress the  For final polishing
block during the process of cutting. Procedure: Heel to Toe movement, Edge first
1. Clean hone with xylene to remove scattered particles of
stones and metal
Other Knives and Blades 2. Cover with lubricant
3. Knife is fitted to its corresponding back, placed on one end
1. Disposable o Widely used now because cheaper; of the hone with cutting edge first
Blades honing and stropping are no longer 4. With cutting knife edge first, the “heel” (handle end) is drawn
common practice; obliquely or diagonally towards the operator on the stone
o coated with polytetrafluoroethylene (for until the “toe” (head portion) is reached.
ease of ribboning) 5. Honing is then continued until all the teeth in the knife edge
o E.g. Magnetic knives in cryostat have been eradicated”
6. Washed with water after using to simply remove the metal
2. Glass Knives/ o For ultrathin microtomes collected during the process
Ralph Knives 7. After honing, wipe off the oil or soap from the knife with
3. Diamond o For resin blocks on ultrathin xylene, then strop it thoroughly……
knives microtomes; brittle and expensive
10-20 strokes per surface for Minot or Plane-wedge knife
4. Safety razor o For partially calcified materials, paraffin • For plane-concave knives, only the concave surface
blades and frozen sections. should be rubbed on the hone
o Easily replaced when dull and produces • Plane-wedge & plane-concave is provided with “backs” to
good tissue sections same as with maintain the correct bevel angle (27-32°) throughout
microtome knives honing
o Unsatisfactory for sections less than
10micra NOTES:
Mechanical Honing
 Utilizes a machine that make use of vibrating frosted slide
plate or wheel driven by electrical motor.
 The knife is pressed against the flat side of a rotating glass
wheel.
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  30 double strokes - given to each side of the knife.
 Advantages: Time-saving; produce well sharpened knives
with uniform bevels
 Disadvantage: Expensive
Stropping
Removal of burrs and polishing of cutting edge
Materials:
 Paddle strop made of horse leather attached to a solid back,
in order to prevent sagging.
- Usually dry thus require oiling
- Vegetable oil (e.g. castor oil) applied on the back of the
horse leather
- Not mineral oil because it tends to blister and destroy
the leather
Procedure: Toe to Heel movement, Edge Last
- The procedure is the reverse of honing
1. The knife is fitted with its appropriate knife back
2. Knife is laid obliquely on the strop and with the cutting
edge behind
3. Edge last is pushed backward and drawn forward
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: both sides are required for
stropping
 Plane – Concave knife: only the concave surface
should be stropped.
TYPES OF TISSUE SECTIONS
1. Paraffin (4-6µm)
• Successive sections will usually stick edge-to-edge (knife)
due to local pressure with each cutting stroke, thereby
forming a ribbon. (remedy: cut slowly)
• Sections are removed in ribbons of ten to allow easy
location of serial sections.
2. Celloidin (10-15µm)
• The blocks are trimmed in the same manner as in paraffin
blocks
• 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 sections do not come off in ribbons and have to
be collected into 70% alcohol immediately.
3. Frozen Sections
• Methods of preparing frozen section
1) Cold knife procedure
2) Cryostat procedure (cold microtome)
(NOTE: More on RAPID TISSUE PROCESSING topic)
FLOTATION/FLOATING-OUT
• For paraffin sections
• Sections are floated out on a water bath set at 45-50°c
(approx. 6-10°C lower than the melting point of the wax used
for embedding the tissue.)
• Flotation Bath
• 5 to 10OC↓MP of Wax
• Inside is specifically colored enamel black
• TSEs flatted after 30sec; removes tse
wrinkling
• Dimensions: d=11in, h=4in, 2L capacity
• Regulated temp. to flatten the sections and prepare them for
mounting into the slides/slider
• Slides (76x25mm, 1-1.2mm thick, frosted)
• 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.
• Adhesives may be used for adhesion of tissue to the slide
(more on ADHESIVES topic)
• Folds or creases sections – may be removed by stretching the
sections gently
• Bubbles – may be teased out beneath the sections by means
of needle
• Selected sections for staining should be fished out in a vertical
position
DRYING THE SLIDES
• Mounted sections are placed in a paraffin oven to dry.
• 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.
References:
1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
(2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
(2015). Basic histopathologic techniques. Metro Manila,
PH: C & E Publishing
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