PRELIMS Flashcards
The machine on which sections are cut
Microtome
Essential parts of a Microtome
1. Knife carrier and Knife
2. Block holder
3. Ratchet feed wheel, pawl, and adjustment screws
Types of Microtome
1. Rocking Microtome
2. Rotary (Minot) Microtome
3. Freezing Microtome
4. Ultra-thin Microtome
5. Sliding or Base Sledge Microtome
The histology technician’s tool of his profession and becomes a personal possession.
Microtome Knife
Each technician should be given at least __ good knife/knives.
2
It is where the tissue is held in position.
Block holder
Adams developed this in 1789.
Sliding or Base Sledge Microtome
Used for actual cutting of tissue sections
Knife carrier and knife
Caldwell Trefall invented this in 1881.
Rocking Microtome
Used to line up the tissue block in proper position with the knife, adjust the proper thickness of the tissue for successive sections
Ratchet feed wheel, pawl and adjustment screws
Principle of the Microtome
A pawl is brought into contact with a ratchet wheel, which is connected to a mill head micrometer screw. This action turns the ratchet wheel, which in turn rotates the screw. By this means the block is moved towards the knife at a predetermined thickness.
It consists of central screw by which the block holder is perforated, attached to it is the food pipe carrying carbon dioxide from a cylinder. A simple lever - operated valve allows the release of rapid intermittent burst of carbon dioxide that freezes the block and the tissue.
Freezing Microtome
T or F: The knife is usually mounted below the block when using a freezing microtome.
Negative. The knife is usually mounted above the block.
Minot invented this in 1885-1886.
Rotary (Minot) Microtome
This is capable of cutting sections required for electron microscope.
Ultra-thin microtome
Ultra-thin Microtome is capable of cutting sections at ____.
0.5 micron
Queckett invented this in 1848.
Freezing microtome
A relatively small length of knife is available to use; it is dangerously placed with blade up.
Rotary (Minot) Microtome
Used in preparation for serial sections. Ribbons of 60-90 sections are obtained with ease.
Rocking microtome
Recommended for cutting extremely hard blocks.
Sliding or Base Sledge Microtome
It is not suitable for cutting large blocks as in the sliding microtome but is convenient for cutting serial sections making it faster for large number of routine blocks.
Rotary (Minot) Microtome
It is the simplest of all microtomes, the tissue block being mounted on the end of a spring poised rocking arm, the knife is held in a horizontal position, the blade face up and slightly inclined towards the block.
Rocking microtome
There are two types of this microtome.
Sliding or Base Sledge Microtome
Its knife consist mainly of selected fragments of broken plate glass. Special diamond knife can be used.
Ultra-thin Microtome
Three basic shapes of the microtome knife
1. Biconcave
2. Plane-concave
3. Plane Wedge
Routinely used for frozen sections and extremely hard specimens embedded in paraffin wax. It is also used for paraffin blocks being cut on the rotary microtome
Plane Wedge
For paraffin sections with a rocking or rotary type of microtome. Can be used on a carbon dioxide freezing microtome
Biconcave
One slide of thin knife is flat and the other is concave. There are 2 degrees of concavity available. The one with the lesser concavity is used for celloidin sections. The more concave side is used for paraffin sections on the sledge, rotary or rocking microtome.
Plane-concave
Care of the microtome knives
- Store the knife in its box when not in use
- Never lay the knife flat in the bench
- Never allow the edge to become badly “nicked”. It is advisable to have a second knife for cutting bone and hard tissue
- The edge of the knife should be “touch up” daily before use
- Always use a back, when required for sharpening. This is not necessary with biconcave knives.
- The knife should be cleaned with xylene or toluene before and after use. Never allow it to become rusty.
- Never lend or borrow a knife.
Has a vibrating frosted glass plate along which the knife is pushed, edge first, held at the correct angle by a knife holder.
Automatic Knife Sharpener
After how many strokes is the blade of the automatic knife sharpener automatically turned over?
4 strokes
A cold chamber, which is kept at a temperature of -20°C. It is used to cut frozen tissue. It has replaced the carbon dioxide freezing microtome for frozen sections.
Cryostat
____ of tissue can be cut using a cryostat
1-4 u
An automatic tissue processor that performs fixation, dehydration, clearing, and paraffin infiltration
Autotechnicon
Used for embedding tissue in an easier way.
Autotechnicon
Autotechnicon parts
- Carrier
- Sample basket - for the specimen
- Receptacle - contains the reagents
- Delay timer - it enables the operator to delay the beginning of the processing cycle for any desired time up to 60 hours
- Timing disc panel- it contains the power switch, selector, pilot light, transparent plastic door with lock and key
- Tissue Embedding Center
Essential parts of the Autotechnicon
1. Freon Refrigeration System - provides the instant cooling of the exchange plate
**2. Paraffin Melting Chamber- **forms a penthouse on the right-hand side of the unit and on the lower left of this chamber is a microswitch dispenser. This chamber maintains an optimal paraffin temperature with an adjustable thermostatic control.
**3. Microscreen **- filters particles or sediments
**4. Micro-switch Dispenser **-Porvides a non-clogging flow of molten paraffin for the casting of molds when the plate is gently pressed
**5. Hot and Cold Orientation Platforms **-For different operations of the embedding procedure.
6. Waste Drawer -Catches excess paraffin
**7. Hot Well - **To pre-heat forceps
Also known as the wax oven designed to store paraffin in its liquid state, maintaining a temperature of 2-5°C higher than the melting point of the wax
Paraffin oven
Designed primarily for use as a micro-sectioned tissue flotation bath.
Flotation water bath
Glass stoppered jars provided at the bottom pad with grooves, usually for fixation and staining.
Coplin jars
Methods in the most common usage for the preparation of specimens for examination
- Sectioning
- Smears
- Dissociation
- Vital Staining
- Autoradiography
The most common method of preparing tissues for histologic examination
Sectioning
12 steps of histopathologic techniques
- Numbering
- Fixation
a. Decalcification- if tissue contains calcium (bone and teeth) - Dehydration
- Clearing
- Infiltration or Impregnation
- Embedding, Casting or MOLDING
- Blocking
- Trimming
- Sectioning or Cutting
- Staining
- Mounting
- Labeling
Advantage of Sectioning
Cell can be studied in their relationship to one another in the tissue and not divorced from their natural background
In what method is the tissue subjected to the 12 steps of histopath techniques?
Can be made by either pressing the cut surface of tissue into slide, an impression smear, spreading material with a wire loop, brushing it between two slides or streaking the specimen into the slide. The preparationg is then fixed, stained, and mounted.
Smears
The tissue is teased with needles into a watchglass containing an indifferent medium such as normal saline and then transferred to a microscpe slide for examination.
Dissociation
Advantage/Disadvatge of Dissociation
A: Cells are examined while still alive.
D: Cells are divored from their normal surroundings.
Used to locate the presence and position of mineral elements in the tissue.
Microincineration
The cells maybe stained by dissociating tissue in a simple staining solution (supravital staining) a method often used to stain reticulocytes, or in the living organism by injecting a dye into the tissue (intravital staining)
Vital Staining
Radioactive isotopes are injected into organs which are then fixed and sectioned
Autoradiography
Type of Examination Requested
- Routine Paraffin Method (most common)
- Papanicolaou Staining method
- Rush Frozen Section
- Decalcification of Bones and Hard Tissues
Enumerate: Simple Fixatives
I. Aldehyde Fixatives
II. Metallic Fixatives
III. Picric Acid fixatives
IV. Acetic Acid fixative
V. Acetone
VI. ALcohol Fixative
VII. Osmium Tetroxide fixative
Enumerate: Aldehyde Fixatives
- Formaldehyde
- Glutaraldehyde
- Paraformaldehyde
A gas produced by the oxidation of methyl alcohol and is soluble in water to the extent of 40% by weight. A powerful reducing agent and used as such in the make up for Helly’s fluid.
Formaldehyde
Usual fixation time if formaldehyde is used.
12-24 hours
Often becomes turbid afer prolonged storage because of the formation of paraformaldehyde which is caused by the polarization of formaldehyde.
Commercial formaldehyde
Methods for removal of Formalin Pigments
1. Kardasewitsch’s Method
2. Lillie’s Method
3. Picric Acid Method
An aldehyde with a MW of 100 or 2 formaldehyde residues linked by a straight 2 Carbon chains
Glutaraldehyde
GLUTARALDEHYDE:
A ___ is best used for small fragments and small needle biopsises, fixed adequately for 2-4hrs in room temp. A _____ is recommended for large tissue not exceeding 4mm in thickness and for 12-24hrs at room temp.
2.5% solution; 4%
A polymer of formaldehyde and is a white powder. It is used in a 4% strength at 4°C.
Paraformaldehyde
Enumerate: Metallic Fixatives
A. Mercuric Chloride
B. Chromate Fixative
C. Lead Fixative
Frequently used in saturated solution. At room temp, its solubility in water is 7%.
Mercuric Chloride
Strong oxiding agents and should not combine with reducing agents such as alcohol and formalin. These are used in 1-2% aqueous solution.
Chromate Fixatives
Used in 4% aqueous solution
Lead Fixative
Usually used in strong or saturated solution. Its solubility is that 1.7g will dissolve in 100ml of distilled water at 15?°C
Picric Acid Fixatives
Chemically, picric acid is __________.
2,4,6-trinitrophenol
Used at ice-cold temperature (-40°C)
Acetic Acid Fixative
Used in ice-cold temp (40°C) and is used only in enzyme studies
Acetone
Used for rapid denaturing and precipitation of proteins by destroying hydrogen and other bonds. Must be used in concentration from 70-100% bec lesser concentrations lyse the cells
Alcohol Fixative
Commonly known as Osmic Acid.
Osmium Tetroxide Fixative
It is pale yellow. It dissolves in water (up to 6% at 20°C) forming a solution which is a strong oxidizing agent
Osmium Tetroxide Fixative
Enumerate: Compound Fixatives
I. Micro-anatomical Fixatives
II. Cytological Fixative
Enumerate: Micro-anatomical Fixatives
A. 10% Formal-saline
B.10% Buffered Formalin
C. Heidenhain’s Susa Solution
D. Formol Sublimate
E. Formol Saline Sublimate
F. Zenker’s Solution
G. Zenker’s Formol (Helly’s Fluid)
H. Bouin’s Solution