Microtomy Flashcards
What is microtomy?
The process of preparing thin slices of biological tissue for microscopic examination of internal structure post embedding.
What is a microtome?
An instrument used to cut biological tissues into very thin, transparent slices for microscopic examination.
List the types of microtomes.
- Retracting
- Vibrating
- Rotary
- Sliding
- Freezing
- Ultramicrotome
Which microtome is most commonly used for sectioning paraffin embedded material?
Rotary microtome.
What are the main components of a microtome?
- Base
- Knife attachment and knife
- Material or tissue holder
How does a rotary microtome operate?
Most operate with a screw feed or by computerized motor, where the block moves up and down, advancing a preset number of micrometers with each revolution of the wheel.
What temperature should sections be floated on a floatation bath?
Approximately 46°C or 10°C below the melting point of the paraffin wax used.
When drying tissue sections, how should they be positioned?
Standing up to let water run down from underneath sections.
What is the recommended drying temperature for slides used in IHC?
Typically 40-50°C for 1 hour.
What factors affect the actual tissue section thickness in a microtome?
- Condition of the microtome
- Quality of knife edge
- Technologist skill
What is the primary maintenance requirement for microtomes?
Lubrication is essential to prevent friction and ensure smooth operation.
What are the advantages of using a freezing microtome?
- May be used to cut paraffin sections
- More portable and less expensive than the cryostat.
What are the disadvantages of a freezing microtome?
- Bulky and dangerous
- Parts must be securely attached
- CO2 bursts may transmit airborne diseases.
What materials are commonly used to make microtome knives?
- Glass
- Diamond
- Sapphire
- Disposable blades made of austenitic stainless or carbon steel.
What is the significance of knife edge quality in microtomy?
A good quality sharp knife can yield satisfactory sections even from poorly fixed tissue.
What are the four profiles of microtome knives?
- Profile A: strongly planoconcave or biconcave
- Profile B: planoconcave
- Profile C: wedge shaped
- Profile D: plane shaped
What is the recommended clearance angle for paraffin sectioning?
3° to 8°.
What are the consequences of an incorrect clearance angle?
- Too obtuse: knife scrapes instead of cuts.
- Too acute: paraffin block is crushed.
What is the bevel angle on a microtome knife?
Formed by upper and lower facets of the knife at 27° to 32°.
What is the purpose of a micrometer setting in a rotary microtome?
To determine the thickness of the tissue section.
What should be done with used knives?
Dispose of in a puncture-proof sharps container that is incinerated.
True or False: Xylene can be used to clean a microtome knife.
False.
What is the main advantage of automated microtomes?
They allow both hands to be free to manipulate ribbons.
What is a disadvantage of the freezing microtome in terms of safety?
The CO2 cylinder must be properly secured to prevent accidents.
What is the wedge angle for microtome knives?
Approximately 15°
What does the angle of tilt represent in microtomy?
The angle between the face of the tissue block and a line bisecting the bevel angle, typically 25°
Define rake angle in the context of microtomy.
The angle formed by the upper cutting facet and a line perpendicular to the tissue block face
What is the slant angle in microtomy?
The angle formed by the lower side of the knife and the surface of the tissue block, ranging from 10° to 14°
What is the function of the knife back?
To maintain a constant bevel when manually sharpening a knife
True or False: Microtomy problems can occur with paraffin, frozen, or celloidin.
True
What happens if the clearance angle or blade tilt is too slight?
Sections that are missed or skipped, alternatively thick and thin, wrinkled and jammed, or lifted from the blade
What are the consequences of having a clearance angle or blade tilt that is too great?
Chatter, microvibration, washboarding, or undulations in sections
What is the recommended clearance angle when installing a sharp trimming knife in the microtome?
3° to 8°
What is the purpose of placing the trimmed block onto a cold surface before cutting?
To harden the face to be cut
What thickness is typically set for cutting most tissue sections?
3 to 5 microns
What can be done to remove wrinkles in sections during the cutting process?
Careful prodding with a camel hair brush or metal probe
What is the purpose of using a warm water bath during sectioning?
To help flatten sections and minimize static electricity
What is the minimum drying time for slides at room temperature?
10 minutes
What causes scored, grooved, or deformed sections?
Bad knife edge or dull knife edge
What is the ‘venetian blind’ effect in microtomy?
It indicates a dull knife
What issue can cause sections to appear mushy?
Poor or insufficient dehydration
What are the four main requirements for successful tissue sectioning?
- Skilled technologist
- Sharp microtome knife
- Proper microtome
- Properly prepared material
What is the ideal temperature for freezing most non-fatty tissue specimens?
-20°C
What is a cryostat?
A stainless steel rotary microtome contained in a refrigerated cabinet
What is the purpose of the antiroll device in a cryostat?
To prevent curling or rolling of frozen sections
Fill in the blank: The embedding medium used in cryostats is called _______.
Optimum Cutting Temperature (OCT)
What is the relationship between temperature and tissue firmness in frozen sectioning?
The tissue firmness is directly related to lipid and water content at any specific temperature
What is the purpose of an antiroll device in cryostats?
Prevents curling or rolling of tissue sections
An alternative to the antiroll device is a fine brush.
What is the freezing compound used in cryostats?
CO2 or Liquid Freon
What does OCT stand for in the context of cryostats?
Optimum Cutting Temperature
What is the function of OCT in tissue cutting?
Prevents tissue from becoming too cold and brittle
What happens if the anti-roll plate is too high above the knife edge?
Frozen block rubs on anti-roll plate
What is a consequence of the anti-roll plate being too warm?
Sticking sections occur
What are the advantages of using cryostats?
- Familiarity with rotary microtome
- No CO2 required for cooling
- Thinner sections possible due to knife sharpness and operator skill
List some disadvantages of cryostats.
- Larger and bulkier than CO2 microtome
- Requires electrical power
- Difficult to clean and disinfect
What is the recommended temperature for storing specimens to prevent dehydration?
-70° C
What is the main use of cryostats?
Rapid diagnosis, often within 15 minutes
What is the first step in the cryostat cutting procedure?
Add a layer of OCT to secure tissue flat
What should be done if tissue sections are thawing during cutting?
Cool the anti-roll plate and knife
True or False: Tissue should be left inside the cryostat for long periods.
False
What is a common problem when using cryostats related to tissue cracking?
Use thinner blocks or freeze slower
What must be done to maintain a clean cryostat environment?
Daily cleaning and proper disposal of tissue debris
What should be done before manipulating the knife or specimen?
Lock the hand-wheel and cover the knife edge with the knife guard
Fill in the blank: Sections will stick to the anti-roll plate if it is too _______.
warm
What is the effect of nicks or debris on the knife edge?
Sections split vertically
What should be done if knife fails to cut?
Check for faulty advance mechanism
How often should cryostats be defrosted?
Frequently
What is the safest way to handle microtome knives?
Always cover the blade and handle with care
What is a potential artifact in microscopic sections from slow freezing?
Ice crystals forming throughout tissue
What is the purpose of using a heat extractant during the cutting procedure?
To further flatten tissue for sectioning
