Microscope is used for the magnification of the tissues and cells
Microtome is used for the cutting of the wax and tissues/cell blocks
There are 3 essential parts of the microtome:
Block Holder (Chuck) – holds the tissue in position.
Knife & Knife Carrier – for actual cutting of tissue sections.
Pawl, Ratcher Feed Wheel & Adjustment Screw – lines up the tissue block in proper position with knife, adjusting thickness of tissues for successive sections.
Microtome is one of the most important instrument in Pathology section
The parts of the microtome should be wiped by using xylol, a clearing agent used for deparaffinization
Rocking (Cambridge) Microtome is invented by Paldwell Tirefall in 1881; it is the “simplest” among types of microtome and is used for cutting serial sections of “large Blocks” of paraffin embedded tissues.
Rotary (Minot) Microtome is invented by Minot 1885-1886; it is the “most Common” type used for both routine and research laboratories and for sectioning paraffin embedded
Sliding Microtome developed by Adams in 1789 is the “most dangerous” type of microtome due to its movable exposed knife; it is used for cutting celloidinor embedded tissues.
The 2 types of sliding microtome are:
Base Sledge Microtome
Standard Sliding Microtome
Freezing Microtome is invented by Queckett in 1848; its is used to cut un-dehydrated tissues in frozen state (for unembedded frozen section) and for unprocessed specimen
Cryostat/Cold Microtome is the most commonly used for “rapid preparation” and a usual type of microtome inside “Rotary Microtome”; it is a refrigerated apparatus used in “fresh tissue microtomy”.
The cryostat/cold microtome has an adjustable thermostat at -5 to - 30°C (average of 20°)
Freshly collected sample is the most used sample type in cryostat/cold microtome
Cryostat/cold microtome is used for rapid diagnostic of cancer
Ultra-thin Microtome is used to produce sections “electron microscopy" where tissues are embedded in “plastic resins”
The Knives used in ultra-thin microtome are Glass (semi-thin) and Diamond Knives (ultra-thin)
The section thickness used in ultra-thin microscope are:
Semi-thin → 0.5-1 micron
Ultra-thin → 500-1200 angstrom (50-120nm)
Plane Concave conventional steel knives with 25mm length are used for base sledge, rotary and rocking microtome where:
Less Concave side → for Celloidin embedded tissue blocks
More Concave side → for Paraffin embedded tissue blocks
Biconcave conventional steel knives with 120mm length are used for rotary microtome where both sides are concave for cutting paraffin embedded sections; it is the most common microtome knives
Plane wedge conventional steel knives with 100mm length are used for base sledge or sliding microtome where both sides are straight; for frozen sections or very hard and tough paraffin embedded tissues.
Honing or hard sharpening of the conventional steel knives is the removal of gross nicks.
It always uses mineral oils.
20-30 double strokes in each direction; but depends on the condition of knife.
Heel to toe direction
There are 3 Types of Hones used:
Belgium yellow – manual sharpening; it gives best results.
Arkansas – gives more polishing effect.
Fine Carborundum – much coarser than the first two; used only for badly nicked knives by either one of the first 2 knife sharpeners.
Stropping or polishing of the conventional steel knives is the removal of burr (formed during honing).
It is done to polish and sharpen the cutting edge.
Toe to heel direction
40-120 double stroke are usually required
Disposable Blades are more commonly used microtome knives where Honing and stropping process is eliminated; it is cheaper to use than conventional steel knives and it is used in Rotary microtome
Magnetic Knives can attach to some blade holders; it is particularly suitable for use in the cryostat
Glass Knives are used for trimming and semi-thin “survey” sectioning of tissue blocks for electron microcopy; with a section thickness of 0.5-1 micron
Diamond Knives is used only for cutting “ultra-thin” sections for Electron microscope and also used in any type of resin block for Electron Microscopy; it is brittle, expensive, but very durable; has a thickness of 500-1200 angstrom
The bevel of the cutting angle of the microtome is 27-32°; the perfect and optimum Cutting Angle is 15°
the Clearance angle used in microtome is 0-15°:
To prevent uneven sections or alternate thin and thick sections, the knife should be inclined with 5-10° clearance angle.
Once above 15°, the angle will not be even
Automatic Tissue Processor only has 4 steps in histopath section:
Fixation
Dehydration (most critical stage)
Clearing
Infiltration
The Advantage of automatic tissue processor is Constant agitation
Wax impregnation is a vacuum embedding apparatus under negative atmospheric pressure inside an embedding oven.
Vacuum embedding gives the fastest result.
Orientation is the most important step in embedding
Floatation Water Bath used to float out and flatten paraffin ribbons; it prevents folding/stretching out the paraffin
Floatation Water Bath is set at a temperature of 45-50°C; approx. 6-10°C lower than melting point of the wax used.
Oven (processes):
Infiltration process → regular at 55-60°C
Drying process → done at 56-60°C (2 hours)
Impregnation → if automatic processor cannot be used
Hot Plate may be used instead of drying oven; it is not recommended since they can cause overheating and there is a risk of dust falling onto the section during drying period.
Forceps (Fine Pointed/Curved) and Squirrel Hair Brush are used in handling sections during cutting, and for removing folds and creases on the sections during “floating out” in water bath.
For clean slides:
For routine work → 76x25mm slides that are 1.0-1.2 thick are preferred.
Frosted end slides are preferred, where the ID number of the section can be inscribed with a pencil.