HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MID-YEAR TERM | MT (UNOFFICIAL NOTES)

TISSUE PROCESSING 4 2. ROTARY (MINOT) MICROTOME

o Invented by Minot in 1885-1886
(EMBEDDING & SECTIONING) o For cutting paraffin embedded tissues
Lecturer: Mrs. Ofelia Racaza
3. SLIDING MICROTOME
EMBEDDING AND MICROTOMY
o Developed by Adams in 1789
You have now your processed tissue after impregnation.
o For cutting celloidin sections
TWO TYPES OF SLIDING MICROTOME
EMBEDDING
BASE-SLEDGE TYPE
DEFINITION
- The chuck or block holder is set on a heavy metal base which
- Also known as or termed as casting or blocking
can be moved backward and forward under the knife during
- a process whereby the impregnated tissue is placed into a
cutting.
precisely arranged position in a mold containing a melted
STANDARD SLIDING MICROTOME
medium which is then allowed to solidify
- The block remains stationary while the knife is moved/slide
- the term embedding medium or embedding media designates
backward or forward during sectioning.
all materials used to infiltrate support or enclose specimens which
are to subsequently cut into thin sections - the most dangerous type due to the movable exposed knife
- the medium we used in infiltration is same in embedding
- generally termed as embedding medium 4. FREEZING MICROTOME
o Invented by Queckett in 1848
ELECTRIC PARAFFIN WAX DISPENSER
o Also known as cryocut, cryostat, or cold microtome
instrument used to melt paraffin wax from solid to liquid state
o Used for cutting unembedded frozen tissue sections
upon heating

PROCEDURE 5. ULTRA-THIN MICROTOME

1. Fill embedding mold with the melted paraffin o Used for cutting ultra-thin sections
2. Pick up now one piece of tissue form paraffin bath using warm
o for electron microscopy
forceps.
3. Place the tissue down to the bottom of the mold parallel to the
bottom. THREE ESSENTIAL PARTS OF MICROTOME
4. Press all sides of the tissue then affix the label on one side of the 1. BLOCK HOLDER
mold. o Where the tissue is held in position
5. Place immediately in ice for solidification. 2. KNIFE CARRIER AND KNIFE
6. Don’t let it solidify in room temp to prevent formation of large o For actual cutting of tissue sections
paraffin crystals and to hasten the solidification. 3. PAWL, RATCHET FEED WHEEL AND ADJUSTMENT SCREWS
What kind of mold do we use in the laboratory? o To line up the tissue block in proper position with the
pop out embedding mold meaning open and close knife
o adjusting the proper thickness of tissue for successive
TYPES OF MOLD
sectioning
(read reference book: GREGORIOS page 194)
§ Leuckhart’s Embedding Mold
§ Compound Embedding Unit TYPES OF MICROTOME KNIVES
§ Plastic Embedding Ring and Base Mold 1. PLANE-CONCAVE KNIFE
§ Disposable Embedding Molds o One side is flat while the other is concave
o Peel-Away TYPES:
o Plastic Ice Trays § LESS CONCAVE - used for cutting celloidin embedded tissue
o Paper Boats
on a sliding microtome
§ MORE CONCAVE - used to cut paraffin section on base-
MICROTOMY
DEFINITION sledge, rotary, or rocking microtome
the process whereby the paraffin embedded tissue block is trimmed
and cut into uniformly thin section to facilitate studies under the 2. PLANE-WEDGE KNIFE
microscope o Both sides are straight
Therefore, you have now your paraffin embedded tissue block, so o Used for cutting paraffin sections
you’re going to trim the four sides and remove excess wax.
3. BICONCAVE KNIFE
MICROTOME o Both sides are concave
the machine we use in cutting
o Used for cutting paraffin sections on rotary microtome
FIVE TYPES OF MICROTOME o Rarely used
(read reference book: GREGORIOS page 204)
1. ROCKING (CAMBRIDGE) MICROTOME
o invented by Paldwell Trefall in 1881
o for cutting serial sections of large blocks of paraffin
embedded tissue

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 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES
MICROTOME KNIFE ANGLES
(read reference book: GREGORIOS page 212)
§ BEVEL ANGLE
o The angle formed between the cutting edges of the
knife
o between 27-32°
§ CLEARANCE ANGLE
o the tilt of the knife ranges from 5 to 10°
o Angle formed between the tissue block and the cutting
edge of the knife between two smooth plane surfaces
(between tissue block and cutting edge of the knife)
The knife that we use now is a disposable knife. However, if you
use the conventional type (the heavy type of knife), it becomes
dull and badly nicked. Hence, you have to sharpen your knives.
TWO STAGES IN SHARPENING THE KNIFE
1. HONING (coarse sharpening)
o Removal of gross nicks and irregularities on the knife
edge
o You’ll know if you have nicks if there are ripped edges
on your tissues
o The direction for honing is from “heel” (handle end) to
“toe” (head portion) – READ BOOK: GREGORIOS pg.
213
o Diagonal stroke; the cutting edge of the knife comes
first (?)
DIFFERENT TYPES OF HONES:
§ Belgium yellow
§ Arkansas stone
§ Fine carborundum
MID-YEAR TERM | MT (UNOFFICIAL NOTES)
TRIMMING
Trim the paraffin block by removing excess wax or excess
wax are cut off (sides, top, bottom of the tissue block) to
1
have a parallel sides
o Paraffin edges - essential to produce paraffin ribbon
Place the trimmed paraffin block in ice beside the
microtome
• Paraffin block at room temperature become soft,
2
so you have to keep it harder by placing it in ice.
PURPOSE: To harden the paraffin block in order to cut tissue
sections
Mount the paraffin block on the block holder
PURPOSE OF BLOCK HOLDER: To hold the tissue in
position during cutting
NOTE:
3
• In mounting, make sure that the paraffin block is
tightly clump to the block holder (If loose, it cannot
cut tissue section)
• Make sure that both upper and lower of edges of
the block holder is parallel to the knife edge
Set the desired micron thickness by turning the knob
Standard thickness: 4-6 micra
Average thickness: 5 micron
4
• using the Pawl, Ratchet Feed Wheel (?) - wait for
the demo; somewhat rapid, even pacing clockwise
rotation of the flywheel (?); purpose is to expose the
tissue
5 Formation of the paraffin ribbon ready for mounting
Mount the tissue section on the glass slide
o clean glass slides, free of dust, holding the edge
6 only (so no fingerprint shown in the slide), then
apply adhesive to let the tissue adhere/stick to the
glass slides

2. STROPPING (fine sharpening) TECHNIQUES IN CUTTING PARAFFIN SECTION

PURPOSE: Trim the tissue block
§ To remove the “burr” and wire edge produced by the stone
Remember in microtomy: Paraffin-embedded tissues
during honing
are trimmed.
§ To polish and sharpen the cutting edge
1 Trimming - removing excess wax to the sides, top and
If the knife becomes dull and blunt, but is free from nicks or teeth, bottom of the tissue block so that the sides are parallel
you can strop it directly (no need of honing). - Parallel edges - essential for ribboning
NOTE: You cannot form a paraffin ribbon if you won’t trim
your paraffin block.
The direction for stropping is from “toe” (head portion) to “heel”
The trimmed tissue block will be chilled and placed in ice
(handle end).

PURPOSE: To harden the paraffin block (since it becomes

REQUIREMENTS TO PRODUCE A GOOD QUALITY TISSUE SECTION
2 soft at room temperature)
1. Skill of the microtomist
• You must always have a container of ice beside the
o Experience is far more essential than reading books
microtome
2. A sharp knife and in good condition • Always from the ice to the microtome
3. Microtome in good condition Mount the chilled paraffin block on the block holder

PURPOSE OF THE BLOCK HOLDER: Where the tissue is

held in position during cutting
3 • make sure that the block is tightly clamped to the
block holder
• If it is loose, you cannot get good tissue sections
(uneven thickness)
• the adjustment screws should be tightened also

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 HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MID-YEAR TERM | MT (UNOFFICIAL NOTES)
• both upper and lower edges of the paraffin block CELLOIDIN SECTIONING
is parallel to the knife - second to paraffin
edge (ASM: the tissue block and the block holder - Celloidin was used before, but now replaced with paraffin
parallel to the knife edge) - In this type of sectioning, the blocks are also trimmed but it
Choose the desired micron thickness of the desired section does not require chilling before cutting.
- Celloidin sections have 5-10 micron thickness, you cannot cut
4
Standard thickness: 4-6 micra (set through the knob) the tissue section and it does not form any ribbon
Average: Set at 5 micron thickness TWO TYPES OF CELLOIDIN IMPREGNATION
Pawl, Ratchet Feed Wheel and Adjustment Screws WET METHOD
• when facing the microtome, the Pawl Ratchet - preferred method for celloidin cutting to avoid dehydration
Feed wheel is on the left side while the hand and shrinkage
5 wheel/fly(?) wheel is on the right side DRY METHOD
• using the Pawl Ratchet Feed Wheel, rotate it to
advance the block holder towards the knife to !! READING ASSIGNMENT !!
expose the tissue (GREGORIOS CHAPTER 13 page 226)
Once the tissue is exposed, rotate the fly (?) wheel in a Faults and problems encountered during tissue processing and
6 clockwise rotation, somewhat rapid or increasing and even during cutting of tissue section, and also their corresponding
pacing (do not attempt to stop) remedies (troubleshooting).
You can now form a paraffin ribbon
• you can place and see the ribbons on the
7
illustration board (black side) since the sections
are white
Once you will have your tissue sections or paraffin ribbon,
these are ready for mounting on the glass slides
• you must have a clean glass slide (free of dust, oily
8 substances, fingerprints)
• hold the glass slide on the edge only
PURPOSE OF GLASS SLIDES: where the tissues are
mounted which will be ready for microscopic study
Apply an adhesive on the glass slide

9 Meyer’s Egg Albumin (commonly used adhesive)

PURPOSE OF ADHESIVE: let the tissue sections adhere or
stick to the slides
Remove the wrinkles and folds on the tissue sections first by
using the teasing needle (stretches the folds on the tissues)

10
TEASING NEEDLE: removes folds from the tissue section
• if there are folds, tissues would be difficult to
interpret
Float the tissues on the tissue flotation bath
• this flattens or spreads the tissue sections
• the temperature should be between 45-50°C, or
6-10°C below the melting point of paraffin wax
• Overheated floatation bath can cause artefactual
separation of tissue sections: meaning, the
11
sections will be ruptured/mabungkag
• once you see the sections already spread out, fish
out the sections (do not let it stay long in the bath)
• Black background purpose: in order to see the
sections which are usually colorless or very light in
color.

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