Microtomy Flashcards
a skilled process that requires
precision and hand - eye coordination combine
with a delicate touch by the experience
histotechnologist/cian
Microtomy
The basic instrument that is capable of cutting a section at a
predetermined thickness by sliding the block into a cutting tool, usually a
steel knife, glass or diamond blade, which is fixed and attached to the
machine.
Microtome
Important parts of microtome
- Block holder
- Knife carrier and knife
- Pawl, ratchet feed wheel and adjustment screws
for actual cutting of tissue sections
Knife carrier and knife
To line up the tissue
block in proper position with the knife, adjusting the proper thickness of the
tissue for successive sections.
Pawl, ratchet feed wheel and adjustment screws
where the tissue is held in position
Block holder
What is the principle of microtome
a spring-balanced teeth or pawl is brought into contact
with, and turns a ratchet feed wheel connected to a
micrometer screw, which is in turn rotated, moving the
tissue block at a predetermined distance towards the
knife for cutting sections at uniform thickness.
Types of microtome
- Rocking microtome
- Rotary microtome
- Sliding microtome
- Freezing microtome
- Cryostat or cold microtome
- Ultrathin microtome
for cutting serial sections of large blocks of
paraffin embedded tissues.
Rocking microtome
for cutting sections for Electron Microscopy.
Ultrathin microtome
for cutting celloidin embedded sections.
Sliding microtome
for cutting frozen section
Cryostat or cold microtome
for cutting paraffin embedded sections
Rotary microtome
-for cutting unembedded frozen sections.
Freezing microtome
Who invented rocking microtome
Paldwell Trefall in 1881
Thickness of rocking microtome
10-12 u
simplest
among the different types of microtome
Rocking (Cambridge) Microtome
Disadvantage of rocking microtome
- restriction in size of tissue block
- difficulty of reorienting the block
Who invented rotary microtome
Minot in 1885-1886
currently the
most common type used for both routine and
research laboratories, especially for sectioning
paraffin-embedded tissues
Rotary microtome
available in
two sizes, has been used to cut small and large
block of paraffin tissues
Rocking microtome
Two types of sliding microtome
- base sledge microtome
- standard sliding microtome
Used for routine and research laboratories
Rotary microtome
Electrically driven rotary microtomes are also now
available and can be ideally used to produce
ribbons for serial sections
Rotary microtome
Thickness of microtome
3-5 um
Who developed sliding microtome
Adams in 1789
Especially recommended for cutting extremely
hard and rough tissue blocks
Sliding microtome
Most dangerous type of microtomedue to the
movable exposed knife
Sliding microtome
Consists of two
movable pillars holding the adjustable knife
clamps, allowing the knife to be set an angle for
cutting celloidin sections
Base sledge microtome
The block
remains stationary while the knife is moved
backward and forward during the process of
sectioning
Standard sliding microtome
Originally designed for cutting sections of
very large blocks (whole brain)
Base sledge microtome
Who invented freezing microtome
Queckett in 1848
The stage for block holder is hollow and perforated
around its perimeter, attached to a reinforced
flexible lead pipe thru which carbon dioxide
passes from a cylinder
Freezing microtome
used to cut undehydrated thin to semi-thin
sections of fresh, frozen tissues
Freezing microtome
Freezing microtome is used in
- rapid diagnosis is required
- histological demonstration of fat is needed
- certain neurological structures are to be studied
- sensitive tissue constituent to be studied is damaged or destroyed by heat
Give the best results and is used almost
universally
Freezing microtome
It consists of a microtome, usually a rotary
microtome, kept inside a cold chamber
Cryostat or cold microtome
Temperature of cryostat
-15 to -30 (ave is -20)
Thickness of cryostat microtome
4 u
means of preparing thin sections of
fresh frozen tissues especially for fluorescent antibody staining
techniques or histochemical enzyme studies
Cryostat microtome
most commonly used for rapid preparation of urgent tissue
biopsies for intraoperative diagnosis.
Cryostat microtome
equipped with a glass or gem grade diamond knife is used to cut
very thin sections (typically 60 to 100 nanometer) of tissue embedded in epoxy resin
ultrathin microtome
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 microtome
Thickness of ultrathin microtome
0.5 to 1 um
Microtome knives
Plane-Concave
Biconcave
Plane wedge
Knives for electron microscope
Glass knives
Diamond knives
Disposable blade
Length of plane-concave
25 mm
Length of biconcave
120 mm
– for celloidin embedded
tissue block on sliding microtome
Less concave
paraffin sections on base-sledge,
rotary, rocking microtome
More concave
For paraffin embedded section on a rotary
microtome
Biconcave
For extremely hard and tough specimen
embedded in paraffin, using a base sledge type or
sliding microtome
Plane - wedge
the angle formed between the
cutting edges normally about
27-32 °
Perfect and optimum angle is obtained when
the sides of the wedge knife are inclined at an
angle of about
15°
Size of glass knives
40x25 cm
Used for trimming and semi-thin sectioning of
tissue block
Glass knives
Used to cut any type of resin block
Diamond knives
Cheaper to use than conventional steel knives
Disposable blades
Brittle and expensive, but very durable
Diamond knives
Disposable blade can cut _____ um thick sections
2-4
removal of gross nicks to remove
blemishes, grinding the cutting edge of the knife
on a stone
Honing
Types of hone
Belgium yellow
Arkansas
Fine carborundum
For manual sharpening
Provides best result
Belgium yellow
Much coarser
Used only for badly nicked knives followed by
either one of the first two knife sharpeners
Fine carborundum
Gives more polishing effect
Arkansas
the process whereby
the “burr” formed during honing is removed and
cutting edge of the knife is polished
Stropping (toe to heel)
Purpose of stropping
Polish and sharpen the cutting edge
Purpose of honing
Remove irregularities from the knife
Process whereby tissues are cut into uniformly
thin slices or “sections” with the aid of a
microtome, to facilitate the studies under the
microscope
Cutting sections
General Types of Tissue Sections
Paraffin section
Celloidin section
Frozen section
For paraffin embedded tissue blocks which may
be cut by _____
rocking and rotary microtome
For celloidin embedded tissues which are usually
cut by means of the
sliding microtome
Frozen Sections
may cut tissues that have been
fixed and frozen with CO2 or for fresh or fixed and
frozen with the cryostat
Factors to be Considered in Sectioning
- Cutting depend upon the type of the tissue
- The size of the block
- The model or the type of the microtome
Sections usually in thickness for routine
histologic procedure
4-6μ
Temperature for waterbath
5-10° below the melting point
Size of slides
76 x 25 mm slides that are 1.0-
1.2 mm
A section is selected for
staining and picked up onto a
clean slide in a vertical
position
Floating Out
Temperature of the water bath during floating out
45 -50°C, approximately 6- 10°C lower than the melting
point of the wax used for
embedding the tissue
substances which can be
smeared on to the slides so
that the sections stick well to
the slides.
ADHESIVES
not necessary for routine
staining
Adhesive
Adhesive is essential for methods that
require exposure of sections to
acids and alkalis
Section can fixed to the slides
37C incubator overnight
Wax oven at 56-60C for 2 hrs
Drying on a hot plate at 45-55 for 30-45 mins
For delicate tissues like CNS or brain 37C for 24 hrs or longer
Why does delicate tissues need to be in low temperature?
To avoid splitting and cracking of section due to excessive heat
Instances when sections may float from the slide and adhesive are necessary
- Urgent cryostat sections to be submitted for
immunocytochemistry - Central nervous system tissues
- Tissues containing blood clot
- Tissues which have been decalcified
- When sections are to be subjected to high temperatures
Common Adhesives Used
- Mayer’s egg albumin
- dried albumin
- gelatin
- starch paste
- plasma
Component of Mayer’s egg albumin
Glycerin
Egg white
Thymol Crystals
Component of dried albumin
Sodium chloride
Dry albumin
Thymol crystals
Component of gelatin
Gelatin 1 gm
Glycerol 15 ml
Phenol crystal 2 gm
dissolved in DW 100 ml
Component of starch paste
Powdered starch
2 drops of hydrochloric acid
Thymol crystals
This is widely used as a section adhesive in
immunohistochemistry
Poly-L-lysine
Concentration of poly-L-lysine
0?1% solution which is further diluted in 1:10 DW
Final dilution of poly-L-lysine
0.01%
APES-coated slides are very useful in cytology,
particularly for
cytospin preparations of
proteinaceous or bloody material
3-aminopropylthriethoxysilane
Advantage of 3-aminopropylthriethoxysilane
can be stored for a long time without losing
their adhesiveness
most commonly used because it is very easy to make, is
convenient, and is relatively inexpensive.
Mayer’s Egg Albumin
Composition of gelatin formaldehyde mixture
- 1% gelatin 5 ml
- 2% formaldehyde 5 ml
