MICROTOMY Flashcards
previously processed tissue / tissue block is trimmed and cut into uniformly thin slices or sections
Microtomy
principle of microtomy
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
when was rocking (cambridge) microtome invented
1881
who invented the rocking (cambridge) microtome
Paldwell Trefall
- the simplest type of microtome for small and large paraffin-embedded blocks
rocking microtome
thickness of rocking microtome
10-12u
one of its disadvantage is, it will not form serial sections
rocking microtome
part of the rocking microtome that carries the block holder
upper arm
part of the rocking microtome that supports the column
lower arm
commonly used microtome for paraffin-embedded sections
rotary microtome
allows production of excellent serial sections
rotary
heavier and more stable microtome with up and down vertical movement which results to perfectly flat planes
rotary
who invented the rotary m
minot
when was the rotary microtome invented
1885-86
thickness of rotary m
4-6u
2 types of sliding m
- standard sliding
- base-sledge
the thickness of sliding m
4-9u
who invented the sliding m
Adams
when was the sliding m invented?
1789
sliding m that consists of 2 movable pillars holding the adjustment knife clamps for hard tissues and large blocks
base sledge
sliding m that is prone to danger
standard sliding
block remains stationary, the microtome moves in a forward and back motion
standard sliding
for hard and tough tissue blocks in all forms of media
base-sledge
for electrom microscope
ultra thin microtome
thickness of ultra-thin m
0.5u
uses fragments of broken plate glass
ultrathin microtome
who invented the freezing microtome
Queckett
when was the freezing microtome invented
1848
for undehydrated tissues in frozen state
freezing microtome
for rapid diagnosis and for fats and tissue constituents that may be damaged
freezing microtome
thickness of freezing m
10-15u
source of cold temp for freezing microtome
carbon dioxide
sections to be focused under compound microtome
- plane-concave
- plane-wedge
- biconcave
for paraffin and celloidin embedded sections
plane-concave
length of plane-concave
25mm
for celloidin-embedded sections and for extremely hard tissues
plane-wedge
length of plane-wedge knife
100mm
for rotary microtome and for paraffin-embedded sections
biconcave
Knives: sections to be focused under EM
- Diamond-edge
- glass knives
resin blocks for UTSM and for EM; knife is expensive
diamond-edge
for clearing and semi-thin sections
glass knives
Used for modern lab, cheaper and convenient, composed of conventional steel knives
Disposable blades
Microtome knives angle
- clearance angle
- wedge angle
- bevel angle
- rake angle
Between the edge of the knife and the tissue block
Clearance angle
Angle of cutting
Wedge angle
Angle of cutting facet
Bevel angle
(CA-BA) - 90°
Rake angle
Angle of CA
5-15°
Angle of WA
15°
Angle of BA
27-32°
Tests for microtome knife shapening
- should cut a paraffin wax block at 2-4 um thickness w/o serrations when examined under the microscope (100x)
- von mhol’s criterion
- will split a hair drawn across it with only their own resistance
Test that confirm shapening of knife is good when there is a Straight narrow, continuous life reflected from the knife edge until the other end if light is focused on the knife
Von Mhol’s criterion
To remove nicks and irregularities
Honing
Hones
- Carborundum
- arkansas
- Belgium yellow
- Belgian black vein
Most expensive hone
Belgium yellow
For rough side of honing
- carborundum
- arkansas
For smooth side of honing
Belgium yellow
T/f the finer the grain the harder the hone
T
Used for final polishing
Diamantine
After repeated sharpening, widened bevel angle (more than 35°)
Factory grinding
T/f hones utilizes oilstones
T
Material for honing
Smooth stones and machine hone
To remove burrs and for final polishing
Stropping
Lubricant for honing
- soapy water, oil (mineral oil, castor oil, clove oil) or xylene
Lubricant for stropping
Vegetable oil
Direction for honing
Heel to toe
Direction for stropping
Toe to heel
Motion and no. Of strokes for honing
-zigzag
- 10-20 single strokes
Motion and no. Of strokes for stropping
-zigzag
- 40-120 double strokes
Flatten tissues in water bath at 10C lower than MP of paraffin wax
Floating
What is used for adhesion
Mayer’s egg albumin
Composition of Mayer’s egg albumin
-egg white
- glycerin
- thymol
Transfer of tissue sections/ribbons on the slide using camel’s hair brush/applicator stick
Fishing out
Correct positioning of the tissue section or ribbon on the slide
Orientation
General steps in fixing sections onto slide
- Floating
- Adhesion
- Fishing out
- Orientation
- Deparaffinization
- Drying sections
- Post- mordantinh
To remove excess paraffin
Deparaffinization
Equipment for deparaffinization
- alcohol lamp
- paraffin oven
Tissue will adhere to glass slide for long duration for staining
Drying sections
Equipment for drying sections
- wax oven
- incubator
-hot plate - alcohol lamp/ bunsen burner
Temp for wax oven for drying sections
56-60 C for 2 hrs
Temp for hot plate for drying sections
45-55C for 30-45 mins (fastest)
To improve the demo of elements in the tissue in staining
Post- mordanting
Secondary fixation(post-chroming) and used primarily as mordant and secondary fixative
Post - mordanting
