MICROTOMY Flashcards
After tissues have been through processing and has been embedded in wax, they are now ready to undergo the process called_____.
microtomy
Microtomy, also known as____ or _____, is the process where tissues are cut into uniformly thin slices to facilitate microscopic studies.
sectioning or cutting
for most paraffin embedded tissues these are sectioned from ___ to ____
4 to 8 microns
The microtome mechanism slowly moves a block into the path of an extremely sharp_____
steel knife
Microtome knives may be generally grouped into four types:
(1)standard thick metal
(2) thin disposable blades
(3) glass knives
(4) diamond knives
These knives allows sharpening to your liking.
Standard thick metal knives
These knives are considered to be expensive.
STMK
In the contrary the______ blades do the same job and probably better for a much lesser cost.
thin disposable blades
Tissue sections that are embedded in plastic material such as methacrylate, araldite, or epon are sectioned with a….
glass or a diamond knife
Tissue sections that are embedded in plastic material such as (3) are sectioned with a glass or a diamond knife.
methacrylate, araldite, or epon
A glass knife can section down to about____
1 micron
For electron microscopy thin sections are preferred and they are sectioned to about _______utilizing a diamond knife.
1/4 of a micron
Sectioning tissues is a real art and takes much skill and practice. Histotechnologists are the artists of the laboratory.
It is important to have a properly____ and ____ block or much artifact can be introduced in the sectioning.
fixed and embedded block
Common artifacts include…
tearing, ripping, holes, folding, etc.
Once sections are cut, they are floated on a_____ that helps remove wrinkles.
Then they are picked up on a____
warm water bath
glass microscopic slide
As a general principle,_____ of the section at the cut line is never advantageous in histology or material sectioning.
excessive bending
Errors of too shallow and too steep an angle are damaging to the specimen. This leads inevitably to the conclusion that the _____should always be set
correct knife angle
Position the lower bevel face____ to the specimen block and the plane of motion.
Then raise the angle slightly above the bevel angle to avoid having the lower bevel face slide over the specimen block and possibly produce friction damage.
The correct knife angle positioning should be consistent regardless of the type of microtome, or any specimen property.
parallel
The ______prevents contact between the knife facet and the face of the block.
clearance angle
The________ is the angle between the two facets that form the cutting edge.
facet or bevel angle
For routine use knives and disposable blades are made with a facet angle of approximately____, but this angle can vary with the blade type and from manufacturer to manufacturer.
Therefore for each blade type the clearance angle must be optimally set.
35°
For rotary microtome knives and blade holders a clearance angle between_______ is recommended.
0° and 15°
kay ma’am - 5°
Problems in sectioning
A. If the ribbon is crooked rather than straight
B. If a ribbon will not form and each section comes off separately
C. If the sections are compressed or folded
D. If the specimen crumbles or falls out of the paraffin section
E. If the ribbon continuously splits or scratches vertically
F. If the ribbon wraps itself around your finger, the knife blade or anything else
Remedies
If the ribbon is crooked rather than straight:
• The upper and lower edges of the block face are not parallel to each other or to the knife edge.
• The knife edge may be uneven. Try another section of the knife edge.
Remedies
If a ribbon will not form and each section comes off separately.
• The blade edge is dull.
• The knife has the wrong tilt.
• The upper and lower edges of the paraffin block face are crumbled or rounded.
Sharpen it.
Adjust it.
Retrim with a sharp razor blade.
Remedies
If the sections are compressed or folded:
• The knife is dull.
• The knife angle is set too close to the vertical.
• The knife is clogged up with paraffin.
• The sections are too thin.
• The paraffin is too soft because the room is warm.
• The paraffin is still contaminated with xylene.
Sharpen it.
Increase the angle.
Clean the edge with chloroform.
Increase the thickness setting.
Pack the blade with ice cubes. Sometimes holding an ice cube against the face of the block helps.
Re-embed.
Remedies
If the specimen crumbles or falls out of the paraffin section :
• The tissue is inadequately dehydrated or cleared.
• The tissue is too hard and compact (e.g., liver).
• The tissue was in the paraffin oven too long at too high a heat.
• The tissue is too hard for a paraffin matrix (e.g., bone, cuticle, heavy plant fibers, or xylem).
Dissolve off the paraffin, rehydrate, and clear.
Soak it in water or 70 % ethanol to soften it.
Throw it away.
Try celloidin embedding.
Remedies
If the ribbon continuously splits or scratches vertically:
• Crystals or dirt particles are caught on the knife edge or on the paraffin face.
• The knife edge is nicked.
Wipe the knife edge carefully and clean it with chloroform.
Use another section of the knife or sharpen it.
Remedies
If the ribbon wraps itself around your finger, the knife blade or anything else:
• The problem is_____, which is often present in very dry room.
• Let _____stand nearby, section in the morning hours or make short ribbons.
static electricity
boiling water
Sectioning
Mechanism
- slow moving of the block into the path of an extremely sharp knife
• allows sharpening; expensive
standard thick metal
• same with (or probably better than) std thick metal; cheaper
thin disposable blades
• plastic-embedded (methacrylate, araldite or epon)
glass knives or diamond knives
diamond knives
• for EM; ultrathin sxns (____)
~0.25 um
Sectioning
Knife Angle
General principle: _____of sxn at the cut line is disadvantageous.
excessive bending
Sectioning
Knife Angle
Errors happen if knife angle is_____
this damages the specimen
too shallow or too steep
- prevents contact between knife facet and face of block
• for rotary microtome -_____
Clearance angle
0° to 15°
- angle between the two facets that form the cutting edge
• knives and disposable blades
Bevel angle (facet)
~35° (variable)
Knife Angle
• Considerations
- incorrect knife angle have various consequences, depending on:
• a specimen type
• fixation and processing qualities
• knife flexibility
