U1 LAB: INSTRUMENTATION Flashcards
Histopathologic Techniques
- Numbering
- Fixation
- Decalcification
- Dehydration
- Clearing
- Impregnation
- Embedding
- Blocking
- Trimming
- Sectioning
- Staining
- Mounting
- Labelling
Numbering is also known as?
Accessioning
This consists of the process: Recording, Logging and Encoding
Numbering
This must be immediately done unless stated by physician.
Fixation
Mineralized specimens include?
bones, deposits in brain (plaques)
This is only done in mineralized specimens.
Decalcification
This step is the removal of water, and uses 70 to 100% absolute ethanol.
Dehydration
This step makes the specimen more clear and transparent, with the use of clearing agents.
Clearing
This makes the specimen miscible to next media.
Clearing agents
Example of Clearing agents
Xylene / Xylol
Impregnation is also known as?
Wax Infiltration
This step refers to the formation of tissue blocks with embedding media.
Embedding
Most common embedding media
Paraffin
Blocking is only done with?
multiple / compound block
This step helps with the tissue block to be more fitted.
Trimming
This step requires the use of microtomy to produce tissue ribbons.
Sectioning
Most common stain in histopath
Hematoxylin & Eosin (H&E)
This step uses a mounting media and coverslip to prevent contamination.
Mounting
This step is a reminder to check if it is properly labelled and stored.
Labelling
Automated Tissue Processors include the following processes:
- fixation
- dehydration
- clearing
- wax impregnation
This tissue processor is characterized by the transfer of tissues, contained within a basket, through a series of stationary reagents arranged in-line or in a circular carousel plan.
carousel-type
Carousel-type processors
What is stationary?
reagents
Carousel-type processors
How many reagent positions can be programmed?
9-10
Carousel-type processors
How many wax positions can be programmed?
2-3
Carousel-type processors
Capacity
30-110 cassettes
T/F: In carousel-type processors, you may reuse the reagents.
True
This is characterized by processing fluids pumped to and from a retort in which the tissues remain stationary.
fluid-type
T/F: Fluid-type processors give out better results.
True
Fluid-type processors
What remains stationary?
tissues
Fluid-type processors
Reagent stations and temperatures
10-12, 30-45C
Fluid-type processors
Wax stations and temperatures
3-4, 48-68C
Fluid-type processors
Capacity
100-300 cassettes
Tissue Embedding Center
This is programmed 2 to 3 degrees higher than melting point of paraffin.
Paraffin Melting Chamber
Tissue Embedding Center
This is pressed to dispense melted paraffin (liquid).
Microswitch
Tissue Embedding Center
This is used for better orientation.
Pre-warming area
Tissue Embedding Center
This is used for the solidifcation of tissue blocks.
Cold orientation platform
This is a basic instrument used to cut sections at a predetermined thickness by sliding the block into a cutting tool which is fixed and attached to medicine.
Microtome
Microtomy is used for what step?
sectioning
Principle of Microtomy
- Spring balance teeth/pawl is brought in contact with ratchet feed wheel
- Ratchet feed wheel is connected to a micrometer screw which rotates
- Moves the tissue block at a predetermined distance towards knife for cutting at uniform thickness
Microtomy
Predetermined distance is dependent on?
thickness
Microtomy
Uniform thickness
5-10um
Microtomy
Specimen holder is also known as?
Chuck
Kinds of Microtome
- Rocking
- Rotary
- Sliding
- Freezing
- Ultrathin
This is the simplest among different types of microtome.
Rocking Microtome
Rocking Microtome
Other name
Cambridge rocking microtome
Rocking Microtome
Invented by?
Paldwell Trefall (1881)
Rocking Microtome
Consists of?
- heavy base
- two arms
Rocking Microtome
Used to cut?
small and large blocks
Rocking Microtome
Not recommended for?
serial sections (tissues are cut in slightly curved planes)
Rocking Microtome
Disadvantages
- Restrictions in size of tissue block that can be cut
- Difficulty of reorienting the block
This is the most common type used for both routine and research laboratories.
Rotary Microtome
Rotary Microtome
Other term
Minot microtome
Rotary Microtome
Invented by?
Minot (1885-1886)
Rotary Microtome
Operated by?
rotation of fly wheel
Rotary Microtome
Rotation of fly wheel causes what motion?
reciprocal motion (knife over block)
Rotary Microtome
Thickness is automatically regulated by?
ratchet feed wheel
Rotary Microtome
Knife and block are brought together by?
upward and vertical motions
Rotary Microtome
Cuts sections in?
perfectly flat plane
Rotary Microtome
Blade is placed in what position?
blade-up
This is the most dangerous type of microtome and is recommended for cutting extremely hard, rough, and large tissues.
Sliding Microtome
Sliding Microtome
Developed by?
Adams (1789)
Kinds of Sliding Microtome
- Base-sledge
- Standard sliding
Sliding Microtome
This kind consists of two movable pillars, and is more stable.
Base-sledge
T/F: Both kinds of sliding microtome is used for cutting celloidin sections.
True
This is an embedding media with a rubbery consistency.
Celloidin
Sliding Microtome
This type is where the knife moves backward and forward, while block remains stationary.
Standard sliding
This microtome is used to cut undehydrated tissues in a frozen state.
Freezing Microtome
Freezing Microtome
Invented by?
Queckett (1848)
Freezing Microtome
Carbon dioxide is reinforced through a?
flexible lead pipe
Freezing Microtome
This is used for freezing the specimen.
Carbon Dioxide
Freezing Microtome
This allows release of rapid intermittent burst of carbon dioxide.
simple lever operated valve
Freezing Microtome
This is used for lowering temperature of knife to facilitate sectioning.
Second cooling device
Freezing Microtome
Indications
- histo demo of fat
- certain neurological studies are to be studied
- sensitive tissue constituents are damaged or destroyed by heat
Freezing Microtome
This consists of a microtome kept inside a cold chamber.
Cryostat
Cryostat
Temperature
-5 to -30C (-20C) by an adjustable thermostat
Cryostat
Capable of freezing tissues within?
2-3 minutes
Cryostat
Can cut sections of ____ with ease
4u
Cryostat
Provides thin sections for?
- fluorescent antibody staining
- histochemical enzyme studies
Cryostat
Most commonly used for?
rapid preparation of urgent tissue biopsies for intraoperative diagnosis
These are used to mount tissues in the Cryotome.
Optimal Cutting Temperature Compound (OCTs)
This is primarily used for cutting tissue sections for electron microscopy.
Ultrathin Microtome
Ultrathin Microtome
Size of tissue sections
0.5 micra
Ultrathin Microtome
Components of knife used
selected fragments of broken plate glass
Ultrathin Microtome
The specimen is small, embedded in plastic, and fixed with?
Osmium tetroxide
Other Kinds of Microtome
This is used for botanical specimens
Vibrating
Other Kinds of Microtome
This is used for efficiency
Laser
Other Kinds of Microtome
This has dual purpose; for routine histopathology and rush frozen sections.
Computerized
Other Kinds of Microtome
- Vibrating
- Hand
- Saw
- Laser
- Computerized
Care of the Microtome
All accumulated paraffin must be brushed away with?
soft brush
Care of the Microtome
After drying the parts, it should be wiped with?
Xylene / Xylol
Care of the Microtome
Movable portions should be?
oiled thoroughly
Trimming and section-cutting are done with a?
microtome knife
This is known as the actual sharp area.
cutting edge
Types of Microtome Knife
One side of the knife is flat, while the other is concave
Plane-concave knife
Plane-concave knife
Length
25mm
Plane-concave knife
Used for?
routine histopath
Types of Microtome Knife
Both sides are concave
Biconcave knife
Biconcave knife
Length
120mm
Biconcave knife
Recommended for?
rotary microtome
Biconcave knife
Used for?
routine histopath
Types of Microtome Knife
Both sides are straight
Plane-wedge knife
Plane-wedge knife
Recommended for?
- frozen sections
- cutting extremely hard and rough specimens
Plane-wedge knife
Used in?
base-sledge or sliding microtome
This is the angle relative to tissue.
Bevel angle
Bevel angle
27-32 degrees
This is the angle that ensures you will be able to cut the block, relative to surface of block.
Clearance angle
Clearance angle
5-10 degrees
This angle refers to the 90 degree angle of the tissue block.
Rake angle
Microtome Knives
These have a sharp cutting edge that can cut thick sections with ease.
Disposable Blades
Disposable Blades
Can cut?
2-4u thick sections
Microtome Knives
This is used for trimming and semi-thin sectioning of tissue blocks for electron microscopy.
Glass knives
Microtome Knives
These are used to cut any type of resin block for electron microscopy, are brittle and expensive.
Diamond knives
Other Equipment for Tissue Sectioning
This allows section to flatten out ribbons
Flotation Water Bath
Flotation Water Bath
Temperature
10C below melting point of paraffin
Flotation Water Bath
What is added to water to reduce surface tension?
small amount of detergent
Other Equipment for Tissue Sectioning
Temperature is set at melting point of wax, 2 to 5 degrees above
Drying oven or hot plate
Other Equipment for Tissue Sectioning
This is also needed in immunohistochemistry
Microwave
Other Equipment for Tissue Sectioning
Needed to handle sections during cutting and removing folds and creases during floating out in water bath.
Forceps, squirrel/camel hairbrush