TRIMMING & MICROTOMY Flashcards
• Removal of excess wax using a knife or cutter after the wax block is removed from the tissue cassette or paper boat.
TRIMMING
TRIMMING
Formation of a_______(ideal shape) and exposure of the tissue surface for ease of sectioning.
truncated pyramid
Allow tissue blocks to fit into the block holder of the microtome.
Trimming
Trimming
___________
• Thicker chunk will be trimmed.
___________
• Small bits or just the edges of your tissue block will be trimmed.
Coarse trimming
Fine trimming
The process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or “sections” to facilitate studies under the microscope
Microtomy
Microtomy
The basic instrument used is a_____
microtome
capable of cutting a section at a predetermined thickness by sliding the block into a cutting tool, usually a (3), which is fixed and attached to the machine.
microtome
steel knife, glass or diamond blade
The microtome consists of three essential parts
Block holder
Knife carrier and knife
Pawl, Ratchet Feed Wheel and Adjustment Screws
Complete ribbons are picked up and transferred to a flotation bath with a (3)
camel hair brush
forceps
fingers
Incomplete sections are_____.
discarded
MICROTOME
● Principle:
Spring-balanced or pawl is brought into contact with, and turns the ratchet feed wheel connected to a micrometer screw, which is, in turn, rotated, moving the tissue black at a predetermined distance towards the knife for cutting sections at a uniform thickness.
5 types of microtome
SRRF CU
Sliding
Rocking
Rotary
Freezing
Cryostat
Ultrathin
– for cutting serial sections of large blocks of paraffin embedded tissues.
Rocking microtome
- for cutting paraffin embedded sections.
Rotary microtome
- for cutting celloidin embedded sections.
Sliding microtome
-for cutting unembedded frozen sections.
Freezing microtome
– for cutting frozen sections
Cryostat or cold microtome
- for cutting sections for Electron Microscopy.
Ultrathin microtome
Rocking microtome aka
Cambridge microtome
simplest among the different types of microtomes.
Rocking microtome
Rocking microtome consists of
Heavy base
2 arms (lower & upper arm)
Rocking microtome
_______ resting on pivots and a supporting column, and attached to the micrometer screw, at the base of which is found the ratchet wheel with feed mechanism.
_______, carrying the block holder on one end by means of a screw, is connected to a lever by a piece of nylon thread.
lower arm
upper arm
Rocking microtome inventor
Paldwell Trefall
1881
Rocking microtome section thickness
10-12um
Knife of rocking microtome
Slightly curved plane
not recommended for serial sections since tissues are cut in slightly curved planes.
Rocking microtome
It is not currently favored by most laboratories because of the restrictions in size of tissue block that can be cut, and the difficulty of reorienting the block.
Rocking microtome
currently the most common type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues.
Rotary microtome
In rotary microtome
knife is fixed in a_____ position
horizontal
Rotary microtome inventor
Minot
1885-86
Rotary microtome
To cut_______ tissues
Both manual and electrically driven models are now available for cutting_____ and ______.
paraffin embedded
ultrathin sections and for cryostat use
Rotary microtome
The cutting angle (tilt) of knife is adjustable, so it can cut harder tissue.
It can cut_______-embedded sections by using a special holder to set the knife obliquely.
celloidin
Rotary microtome section thickness
3 and 5 µm using paraffin wax
It is heavier and more stable than the rocking microtome, is more complex in design and construction
Rotary microtome
Rotary microtome
(A heavier knife is used, so there is_____)
Produces serial sections
less vibration
more expensive
The knife is placed in a blade-up position and is therefore relatively dangerous.
Rotary microtome
knife can be set obliquely for celloidin sections or straight for large refractory paraffin blocks, cutting both large and small tissues with ease;
Sliding microtome
slow but very steady motion is therefore required to manipulate the instrument.
Sliding microtome
It is especially recommended for cutting extremely hard and rough tissue blocks.
Sliding microtome
Sliding microtome inventor
Adams in 1789
most dangerous type of microtome due to the movable exposed knife.
Sliding microtome
Two types of sliding microtome
Base-Sledge Microtome
Standard Sliding microtome
-› electrically driven and ideal for resin-embedded decalcified bone.
Base-Sledge Microtome
-> knife is moving; the most dangerous type of microtome because of the exposed, movable blade.
Standard Sliding Microtome
• Both Microtome, the knife can be set obliquely for celloidin or straight large paraffin.
• Recommended for cutting “extremely” hard and rough tissue blocks.
Sliding microtome
True or False
Sliding microtome
Cannot make tissue ribbons, only tissue sections.
True
It was originally designed for cutting sections of very large blocks (whole brain).
Base sledge microtome
Sections are cut in a perfectly flat plane, thereby making excellent serial tissue sections.
It is comparatively heavier and more stable than the ordinary sliding microtome.
The angle of the knife is adjustable
Base sledge microtome
favored in laboratories where very hard tissue or large blocks are usually sectioned.
suited for sectioning specimens embedded in all forms of media, especially for cutting sections from tough tissue blocks which may offer great resistance to the knife
Base sledge microtome
knife used is long (24 cm), hence it requires less honing
Base sledge microtome
FREEZING MICROTOME
• Invented by:
Quickett (1848)
The stage for block holder is hollow and perforated around its perimeter
Freezing microtome
The microtome is firmly clamped on to the edge of the bench for use, or mounted on especially constructed shelf
Freezing microtome
Freezing agent of freezing microtome
Freezing agent: CO2
Release into the stage & will evenly freeze block holder & tissue.
used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances when rapid diagnosis is required
Freezing microtome
For fats & tissue
constituents that may be damaged
Freezing microtome
For rapid diagnosis in the processing of neurological structures & heat sensitive tissue constituents
Freezing microtome
Freezing microtome section size for neurological tissues
10-15 um (for neurological tissues)
Although other microtomes can be modified for cutting frozen section, this type will give the best results and is used almost universally.
Freezing microtome
the knife is moved whilst the tissue block remains static, same as sliding microtome.
Freezing microtome
Cryostat aka
Cold microtome
True or False
Cryostat is more common than freezing microtome.
True
Used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis (STAT).
Cryostat
Cryostat
• Chamber:
• Average:
-5 to -30°C
-20 °C
Cryostat
Thermostat:
Capable of freezing fresh tissue within…
2-3 minutes
It is often housed in the frozen section room close to the operating room to allow direct consultation between surgeon and pathologist
Cryostat
Sections are usually transferred directly from the microtome knife to a slide or cover glass, all of which are maintained at a low temperature
Cryostat
The cryostat provides a means of preparing thin sections of fresh frozen tissues especially for _____ or _____
fluorescent antibody staining techniques
histochemical enzyme studies.
Cryostat
cutting sections of…
4 µ
• All the controls to the microtome are operated from the outside of the refrigerated cabinet.
Cryostat
Ultrathin microtome
______(epoxy resin)
semi-thin - generally______
60 to 100 nm
0.5 to 1 µm
Ultrathin microtome
used to cut very thin sections of tissue embedded in____
epoxy resin
equipped with a glass or gem grade diamond knife
Ultrathin microtome
Ultrathin microtome
Fixate in
Osmium tetroxide
Embedding medium of ultrathin microtome
plastic
Ultrathin microtome
These semi-thin sections are generally 0.5 to 1 µm thick and are mounted on a glass slide and stained to locate areas of interest under a _____prior to thin sectioning for the TEM.
Thin sectioning for the TEM is often done with a____knife
light microscope
gem quality diamond knife.
Care
After sectioning, all the accumulated paraffin and small pieces of tissues must be brushed away with a ______and not allowed to stay in the microtome, since this may later interfere with the cutting of tissue blocks
soft brush
-> may also be used for cleaning some parts of the microtome.
Xylene
Care
After carefully drying the machine and knife holder, the parts should be wiped with______.
Prolonged and continuous application of the painted parts with____ should, however, be avoided since this reagent is capable of removing the paint.
xylol
xylene
Care
Movable portions should be____ thoroughly to prevent rusting
oiled
The microtome must always be_____ when not in use, to prevent accumulation of dust and other dirt which may later on interfere with the normal sectioning of tissues.
covered
Have the instrument inspected at least______ by a qualified service technician.
once a year
Safety
Use____ or ____ instead of fingers to pick up sections or wax fragments from blade or block face.
forceps or brush
Use________ when changing blocks. The knife or blade should be removed from the microtome when the instrument is left unattended or when cleaning the instrument.
hand wheel lock
