EMBEDDING - TRIMMING - SECTIONING - MICROTOME Flashcards
Aka: Casting/Blocking
EMBEDDING
● Process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify
EMBEDDING
○ Process by which a tissue is arranged in precise positions in the mold during embedding,
■ On the microtome before cutting and on the slide before staining
● Orientation
The surface of the section to be cut should be placed parallel to the bottom of the mold in which it is oriented
EMBEDDING
Temperature of melted paraffin used for embedding
5-10℃ above its Melting Point
What is the other name of “Embedding”?
According to Gregorios it is otherwise
known as “Casting” or “Blocking”
To solidify embedded tissue
Cooled rapidly in a ref (-5℃) or immersed in cold water
STEPS IN EMBEDDING
- Removed the tissue from the cassette
- Fill the mold with wax
- Orientate the tissue
● Process of removing excess wax after embedding.
TRIMMING
is performed in the paraffin oven.
Embedding
● The excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting.
● Knife/Blade may be used.
TRIMMING
Correct shape of the block:
Four-sided prism or Truncated pyramid
● Also known as cutting or microtomy
SECTIONING
● The process by which a processed tissue is cut into
uniformly thin slices (sections) to facilitate studies under the microscope.
○ Sections (Gregorio’s) or Ribbon
SECTIONING
Routine tissue microtomy
3 - 5 u
Frozen sections
10 - 15 u
Very thin sections
- For electron microscopy
60 - 100 nm
Semi-thin sections
- For electron microscopy
0.5 - 1 um
● For cutting serial sections of large blocks of paraffin-embedded tissues
● The simplest microtome
Rocking Microtome
Inventor of Rocking Microtome
Paldwell Trefall in 1881
Disadvantage:
● Difficulty in re-orienting the block
Rocking Microtome
● For cutting paraffin-embedded
sections
Rotary / Minot Microtome
Inventor of Rotary / Minot Microtome
Minot in 1885-1886
● The most common microtome used today especially for paraffin-embedded tissues
Rotary / Minot Microtome
Some microtomes have digital
indicators to adjust the thinness of the sections to be cut.
Rotary / Minot Microtome
● For cutting celloidin-embedded
sections
Sliding Microtome
Inventor of Sliding Microtome
Adams in 1789
The most dangerous type due to
movable exposed knife
Sliding Microtome
2 types of Sliding Microtome:
- Base-Sledge
- Standard Sliding Microtome
can be used for all types of media
- Block holder: moving
- Knife: stationary
Base-Sledge
- Block holder: stationary
- Knife: moving
Standard Sliding Microtome
It is another type of microtome
Rotary Rocking Microtome
Disadvantage:
● Sections are liable to disintegrate
Vibratome
● Used for unfixed, unfrozen specimen sectioning for enzyme demonstrations
Vibratome
● For cutting sections for electron
microscopy
Ultrathin Microtome
Temperature: Ultrathin Microtome
0-4 degree celsius
Impregnating medium of Ultrathin Microtome
Plastic or resin impregnating media
Uses diamond knives or broken plate glass
○ Broken plate glass is only
used as an ALTERNATIVE to
diamond knives
Ultrathin Microtome
● For cutting of unembedded frozen sections
Freezing Microtome
Plane Concave
Usual Length:
Description:
25 mm
One side
● flat
Other side
● concave
● Specimen is small, fixed in osmium tetroxide, embedded in plastic
Ultrathin Microtome
Among the two types of sliding microtome, which is the most dangerous type?
Standard sliding microtome is the most dangerous type because of the exposed movable knife
Inventor of Freezing Microtome
Queckett in 1848
Plane Wedge
Usual Length:
Description:
100 mm
Both sides
● straight
Biconcave
Usual Length:
Description:
120 mm
Both sides
● concave
● How far the knife is from the
tissue block
Clearance Angle
Clearance Angle
0 to 15 degrees
Bevel Angle
27 to 32 degrees
● Types of Hones (3)
○ Belgium Yellow
○ Fine Carborundum
○ Arkansas
● Removal of gross nicks
Honing
gives the best honing result
Belgium Yellow
● Knife direction:
○ Heel to toe
Honing
● Removal of burr
Stropping
● Knife direction:
○ Toe to heel
Stropping
● Instrument:
○ Paddle strop
Stropping
made up of horse leather
Paddle strop
TEMPERATURE OF FLOATATION WATER BATH (2)
● 45-50 degrees Celsius
● 6-10 degrees Celsius LOWER than the melting point of wax
Why LOWER than the melting point of wax
If the temperature is higher than the melting point, the ribbons will melt, and the tissues will submerge to the bottom
Electron Microscopy
0-10°C
Fixation Surgical Specimen
25°C
Paraffin Oven for Impregnation
55-60°C or 2-5°C ABOVE the melting point of paraffin wax
Embedding
5-10°C ABOVE the melting point of paraffin wax
Microwave Fixation
45-55°C
Most common adhesive agent
Mayer’s Egg Albumen
Components of Mayer’s Egg Albumen (2)
○ Egg white/Albumen
○ Crystals of Thymol
to prevent growth of molds
Crystals of Thymol
Used to enhance tissue adherence to slides
Dried Albumin
Commonly used for coating slides
to improve tissue section adhesion
Gelatin
Provides strong adhesion and
prevents tissue loss during staining
Gelatin-Form aldehyde Mixture
Simple and effective adhesive for
mounting specimens
Starch paste
Effective natural adhesive agent for tissue sections
Plasma
Enhances cell attachment,
particularly useful for immunohistochemistry
Poly-L-Lysine
3-APES meaning
3-aminopropyltriethoxysilane
● APES-coated slides
○ very useful in cytology, particularly for cytospin
preparations of proteinaceous or bloody material
3-APES
(3-aminopropyltriethoxysilane)
(2) STAGES OF KNIFE SHARPENING
(1) Honing
(2) Stropping
