EMBEDDING & MICROTOMY HISTOPATH LAB

Embedding

process by which impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify

Orientation, Molding, Blocking, Casting, Trimming

5 Substeps in Embedding

Orientation

precise positioning of the tissue section inside the molder

Molding

provides shape to the tissue sample

Blocking

rapid solidification of tissue block

Casting

removal of tissue block from molder

Trimming

removal of excess paraffin wax from the mold

Leuckhart’s Embedding Mold

w/ 2 L-shaped strips of heavy metal arranged on a flat metal plate

Compound Embedding Unit

made up of series of interlocking plates resting on a flat metal base

Plastic Embedding Rings and Base Mold

w/ special stainless steel base mold fitted with a plastic embedding rings

Plastic Embedding Rings and Base mold

Does not require the removal of the block from the mold

Disposable Embedding Molds

Peel away, Plastic ice trays, Paper boats

Peel away

gives perfect even blocking without trimming

Plastic ice trays

may be recommended for busy routine laboratories

Paper boats

normally utilized for embedding celloidin blocks

Different Types of Blocking Out Molds

Leuckhart’s Embedding Mold

Compound Embedding Unit

Plastic Embedding Rings and Base Mold

Disposable Embedding Mold

Celloidin or Nitrocellulose Method

Double Embedding Method

Plastic (Resin) Embedding

Microtomy

Process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or sections to facilitate study under the microscope

Essential Parts of the Microtome

Block holder

Knife Carrier and Knife

Pawl, Ratchet Feed Wheel and Adjustment Screws

Block holder

where the tissue is held in position

Knife Carrier and Knife

for actual cutting tissue sections

Pawl, Ratchet Feed Wheel and Adjustment Screws

to line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections

Parts of the Microtome

Base,

Cassette or Block holder

Block adjustment screw

Knife holder

Blade clamps

Angle of tilt adjustment

Thickness gauge

Operating handle

Face plate

Waste tray

Types of Microtome

Rocking Microtome

Rotary Microtome

Sliding Microtome

Ultrathin Microtome

Freezing Microtome

Cold Microtome

Rocking Microtome

aka Cambridge microtome

Rocking Microtome

Invented by Paldwell Trefall (1881)

Paldwell Trefall

Who invented Rocking microtome?

Rocking Microtome

Simplest among the different types of microtome

10-12 um

Thickness of tissue sections in Rocking Microtome

Rocking Microtome

Used to cut small and large blocks of paraffin tissues

Rocking Microtome

Not recommended for serial sections

Rocking Microtome

Not currently favored by most laboratories because of the restrictions in size of tissue block that can be cut, and the difficulty of reorienting the block

Minot

Who invented Rotary Microtome?

Rotary Microtome

For cutting routine paraffin embedded tissues and research laboratory uses

Rotary Microtome

Currently the most common type being used

4-6 um

Thickness of tissue sections in Rotary Microtome

Adams

Developed the Sliding Microtome

Sliding Microtome

most dangerous microtome

10-30 um

Thickness of tissue sections in Sliding Microtome

Base-Sledge and Standard Sliding

2 Types of Sliding Microtome

Base-Sledge

the moving part is the chuck or block holder, the knife remains in its place

Base-Sledge

used for very hard tissue or large blocks

Base-Sledge

modern models are ideal for resin-embedded decalcified bone

Standard Sliding

the block remains stationary while the knife is moved backward and forward

Standard Sliding

used for cutting celloidin-embedded tissue blocks

Ultrathin Microtome

is used to cut very thin sections of tissue emedded in epoxy resin

60 to 100 nanometer

Thickness of section in Ultrathin Microtome if glass or gem grade diamond knife

0.5 - 1 um

Thickness of section in Ultrathin Microtome if glass knife or industrial knife

Ultrathin Microtome

Sections are examined with a transmission electron microscope (TEM)

Queckett

Invented the Freezing Microtome

Freezing microtome

Uses intermittent bursts of carbon dioxide which will freeze the block holder and the tissue evenly

Freezing microtome

Is used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances when rapid diagnosis is required

Freezing microtome

Used when histological demonstration of fat is needed and neurological structures

Freezing Microtome

Also used when sensitive tissue constituents to be studied are damaged or destroyed by heat

Cold microtome

aka cryostat

Cold microtome

is a refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning

Cold microtome

usually consists of a rotary microtome kept inside a cold chamber set at a temperature between -5 to -30 degC (average of -20 degC)

4 um

Thickness of section in Cold Microtome

Cold microtome

used for fluorescent antibody testing techniques or histochemical enzyme studies

Cold microtome

commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis

Microtome knives

Plane-Concave

Biconcave Knife

Plane-Wedge

Disposable blades

2 types of disposable blades

Glass knives

Diamond knives

Plane concave

usually 25mm in length

Flat surface in plane concave

cutting celloidin-embedded tissue blocks on a sliding microtome

Concave side in plane concave

cutting paraffin sections on base-sledge, rotary or rocking microtome

biconcave

usually 120mm in length

biconcave

Recommended for cutting paraffin-embedded sections on a rotary microtome

Plane wedge Knife

usually 100mm in length

Plane-wedge

Recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin blocks, using a base-sledge type or sliding microtome

Disposable blades

Cheaper to use than conventional steel knives

Disposable knives

Have a sharp cutting edge that can cut 2-4um thick sections with ease

Glass Knives

Generally used for trimming and semi-thin sectioning of tissue blocks for electron microscopy (EM)

Diamond Knives

Used to cut any type of resin block for electron y

Microtome Angles

Bevel Angle

Clearance Angle

Wedge Angle

Bevel Angle

formed between cutting edges

bevel angle

27-32 degrees

Clearance angle

Formed between SURFACE of the BLOCK and CUTTING EDGE of KNIFE

Clearance angle

0-15 degrees

Wedge angle

Formed by the SIDES of WEDGE KNIFE

Wedge Angle

14-15 degrees

Honing

Grinding the cutting edge to acquire even size edge

removal of gross nicks

Purpose of Honing

Honing

Heel to toe movement

10-20 or 20-30 stroke

# of strokes in honing

Belgium yellow, Arkansas, Fine Carborundum

3 Hones or Oil Stones

Belgium Yellow

gives BEST results

Arkansas

with more polishing effects

Fine Carborundum

for badly nicked knives

Stropping

Polishing and sharpening the cutting edge

Removal of Burrs

Purpose of stropping

Stropping

Toe to heel movement

40-120 double strokes

# of strokes in Stropping

Stropping

Uses paddle strop made of HORSE LEATHER that are usually treated with VEGETABLE OIL or CASTOR OIL at the back and not the surface

Sectioning

Is a process whereby tissues are cut into uniformly thin slices or sections with the aid of a microtome, to facilitate the studies under the microscope.

Paraffin Sections, Celloidin Sections, Frozen Sections

3 general types of tissue sections

Paraffin Sections

for paraffin embedded tissue blocks which may be cut by rocking and rotary microtome

Celloidin Sections

for celloidin embedded tissues which are usually cut by means of the sliding microtome

Frozen sections

for tissues that have been fixed and frozen with CO2 or for fresh or fixed tissues frozen with the cryostat

Flotation

allows the expansion of the section to its original dimensions and ensure that it is completely flat

5-10 degC below the melting point of the wax

Temperature for waterbath

Fishing out

process of removal of ribbons from the water bath, must be done vertically