MODULE 4 - Sectioning Flashcards
Involves cutting of tissues into thin slices
Sectioning
Size of paraffin sections
4-6 um
Size of celloidin sections
10-15 um
Size of frozen sections
4 um
Disadvantage of using cambridge/rocking microtome
Difficult in re-orienting tissues and restriction in tissue size of blocks
Cambridge/rocking microtome is made by
Trekfall
Most simple microtome
Cambridge/Rocking
Cambridge/Rocking microtome is made for
serial sections of large paraffin blocks
Thickness produced when using cambridge/rocking microtome?
10-12 micra
Routinely and most common microtome
Rotary
Rotary microtome is made by
Minot
Thickness produced when using rotary microtome
4-6 micra
Rotary microtome is used for
paraffin embedded tissues
Sliding microtome is made by
Adams
The most dangerous microtome and for what reason
Sliding microtome because of exposed knife
Example of wax used in sliding microtome
Ester wax
Sliding microtome is used for
celloidin embedded tissues and extremely hard tissues
Thickness produced when using sliding microtome
7-9 micra
2 types of sliding microtome
- Base sledge
2. Standard sliding
Most dangerous type of sliding microtome
Standard microtome
Freezing/cold knife uses
intermittent burst of CO2 to immediately harden block holder and tissues without prior fixations.
Freezing/cold knife has a
second cooling device to lower temp of knife
Freezing/cold knife is used for
frozen sections and heat-sensitive structures
Freezing/cold knife is used for demonstration of
fats and neurological structures
Freezing/cold microtome was invented by
Queckette
Dew line is
point of sections in which it can be cut at 10um
thickness and tissue block produced using freezing/cold knife microtome
thickness: 10-15 um
block: 3-5 mm
Type of microtome enclosed in a chamber and is primarily composed of rotary microtome
Cryostat/cold microtome
Cryostat/cold microtome is used for
fresh frozen tissues for fluorescent antibody staining and histochemical enzyme studies
Temperature used in cryostat/cold microtome
-5 to -30 degC
average -20 degC
Microtome used when tissues are usually embedded in plastic
ultrathin microtome
ultrathin microtome is used for
EM
thickness of tissues when using ultrathin microtome
0.5 um
process of removing excess paraffin wax from tissues once properly
fixed on the slide
DEPARAFFINIZATION
METHODS of DEPARAFFINIZATION
- Passing of slide over alcohol lamp
- Immersing slide in xylene
- Putting it on oven (55-60 degC)
Microtome knives that is 25mm long
Plane concave
Microtome knives that is 120mm long
Biconcave
Microtome knives that is 100mm long
Plane wedge
Plane concave flat side is used for?
concave side is for?
Flat- celloidin
Concave- paraffin
Biconcave is used for
paraffin embedded and rotary
Plane wedge is for
frozen sections and extremely hard tissues
Bevel angle is
angle formed between the cutting edges 27-32 deg
Clearance angle is also known as
Tilting/inclination angle
Clearance angle is
angle formed between surface of blocks and cutting edge of knife @0-15 deg but in fixation it is 5-10 deg to avoid uneven sections (alternate thin and thick)
In Clearance angle >10 indicates and <10 indicates
> 10 - low thickness
<10 - high thickness
Wedge angle is
angle formed in sides of wedge knife 14-15 deg
Thickness produced by disposable blades
2-4 um
Glass and diamond knives are used for
EM
Purpose of honing
to remove nicks
Honing uses what movement
heel to toe movement, edge first 20-30 times
Honing uses
hones/oil stones
lubricant
Oil stones used in honing
Belgium yellow
Arkansas
Fine carborundum
Lubricants used in honing
soapy water mineral oil clove oil xylene liquid paraffin
Belgium yellow
gives best result
Arkansas
gives more polishing effect than belgium yellow
fine carborundum
for badly nicked knives, has coarser bite
Alternatives for oil stones
Glassplates and diamantine
What composes glass plates
powdered aluminum oxide with water = paste = ABRASIVE
Diamantine is
for final polishing
Polishing and sharpening of knife
stropping
purpose of stropping
to remove burrss and other deformities formed by honing
stropping is done with what movement and how many strokes
toe to heel, edge last 40-120 double strokes
Strapping uses _____ made of _______
paddle strop, horse leather
Strops are treated with
oil
In stropping do not use ______ because it can ________
lubricant, mineral oil - blister and damage leather
Purpose of floatation water bath
thermostatically controlled bath to remove folds and wrinkles to flatten ribbons
Temperature of floatation water bath when in use
6-10 degC lower than wax melting point
specifically, 45-50 degC
Product of section
thin slices , ribbons
what is fishing out
removal of ribbon in float out bath
3 methods for Drying of slides
- leave in incubator @37 degC overnight
- put in oven @50-60 degC for 2 hours
- in a hot plate @45-55 degC for 30-45 mins
This is used to promote attachment to slides and prevent detachment
adhesives
Routine tissue adhesive
Mayer’s egg albumin
Mayer’s egg albumin is composed of
Egg white + glycerin + thymol crystals
Thymol crystals is used to
prevent growth of molds
Poly-L-Lysine is used for
immunohistochemistry
APES (3-aminopropylthriethoxysilane) is used for
cytology
With strong adhesive property
sodium silicate
Adhesive that is a commercial syrup
sodium silicate
Enumerate methods of freezing and their details
Liquid nitrogen- most rapid, for histochem Isopentane- liquid at RT Aerosol sprays (cryokwik) CO2- freezing microtome Freon 2.2- high thermal conductivity
Microtome parts and their details
- Block holder/chuck- where tissues are held in position
- Knife carrier/knife- disposable knives, for actual cutting
- Rotating/flying wheel- done mechanically to start the cutting process
- Pawl, ratched feed wheel, adjustment screw- lines up tissues in proper positions with knife
A microtome that is theoretically not recommended for serial sections since tissues are cut in slightly curved planes.
Cambridge/rocking microtome
Typically, sections are cut between ___________ µm using paraffin wax for diagnostic histology
3 and 5 um
It was originally designed for cutting sections of very large blocks (whole brain).
Sliding microtome
Microtomes used for serial sections (book)
Rotary
Sliding- basic sledge
It is used to cut undehydrated thin to semi-thin sections of fresh, frozen
tissues
Freezing/cold knife
capable of freezing fresh tissues within 2-3 minutes, and cutting sections of 4 µ with ease.
The Cryostat or Cold Microtome
It is most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis.
The Cryostat or Cold Microtome
It is often housed in the frozen section room close to the operating room to allow direct consultation between surgeon and pathologist.
The Cryostat or Cold Microtome
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
Thin sectioning for the TEM is often done with a
gem quality diamond knife
After sectioning, all the accumulated paraffin and small pieces of tissues
must be brushed away with a soft brush and not allowed to stay in the
microtome, since this may later interfere with the cutting of tissue blocks.
TRUE OR FALSE
True
After carefully drying the machine and knife holder, the parts should be
wiped with
xylol
Prolonged and continuous application of the painted parts with xylene will
remove the paint
Movable portions should not be oiled to prevent rusting
TRUE OR FALSE
False
The microtome must always be covered when not in use
TRUE OR FALSE
True
The microtome should be placed on an unstable bench, away from air drafts, doorways and passing staff.
TRUE OR FALSE
False
Any air movement from air conditioners or other causes will not affect section handling
TRUE OR FALSE
False
No fluid must enter the inside of the instrument during cleaning.
TRUE OR FALSE
True
Have the instrument inspected at least __________ by a qualified service technician.
once a year
Microtome that uses plane concave
rotary or rocking microtome
Each knife should have its own corresponding back which should be interchanged with another, to keep the bevel angle.
TRUE OR FALSE
False
Too soft cutting edges are likely to
dull easily
too hard edges are likely to produce
nicks or jagged edges and irregularities on the knife edge
A good cutting edge must be able to cut good sections from a paraffin wax block about
2-3 microns thick
may be used for partially calcified materials, paraffin and frozen sections.
Safety razor blades
Theoretically, the perfect and optimum cutting angle is obtained when the sides of the wedge knife are inclined at an angle of about
15 deg
The degree of sharpness is __________ to the fineness of the abrasive used in sharpening
proportional
Hones for manual sharpening when cutting edge has been rendered blunt or nicked
belgium yellow
The surface of the hone is wiped clean with a soft cloth moistened with ______ in order to remove the scattered small particles of stones and metal.
xylene
In the case of the Minot or plane-wedge knife, the knife is turned over so as to sharpen the other surface every
I0-20 strokes
Speed in stropping should be avoided
True or False
True
Leather strops are usually dry and require oiling before they are used.
Strops are usually treated with
vegetable oil (castor)
The strop should not be used for at least ________ after treatment.
24-48 hrs
generally used for trimming and semi-thin sectioning of tissue blocks for electron microscopy.
Glass Knives
used to cut any type of resin block for electron microscopy.
diamond knives
used to cut any type of resin block for electron microscopy.
Diamond Knives
These tools are needed for handling sections during cutting, and for removing folds and creases on the sections during “floating out” in water bath.
Forceps (fine pointed or curved) and squirrel hair brush
The quality of sections cut on a microtome suffer badly from these several (avoidable) causes.
Fecal material and hair
Coarse facing is done on the microtome at approximately
30 microns at a time
Cooling both the tissue and the wax will give them a similar consistency, and make sectioning easier
TRUE or FALSE
true
In fine trimming, the knife is usually tilted at
0-15 deg
provides better support for the harder elements in a specimen allowing thinner sections to be obtained.
cold wax
Generally a fast, uniform cutting stroke produces the best results and the least compression.
TRUE or FALSE
False
Do not stop and restart during a cutting stroke as this will produce bands of different thickness across the section.
TRUE OR FALSE
True
Sections should not be left on the water bath for a long time (30 seconds will be enough)
to avoid undue expansion and distortion of tissue.
When flattening out sections, 20% alcohol will produce
convection current
expand the section to its original dimensions and ensure
that it is completely flat.
floatation
not recommended because they can cause overheating and there is a risk of dust falling onto the section during the drying period.
hot plate
Excessive heat can cause
boiling of water droplets underneath the section which will cause damage
Dry sections for between
5 and 30 minutes
Staining of serial sections should never be attempted unless they are completely dried.
TRUE OR FALSE
True
charged slides or an adhesive must be used in
antigen retrieval (IHC) enzyme pretreatment (ISH) prolonged incubation steps
Extended storage (usually more than 3 days) of unstained formalin-fixed paraffin embedded slides will cause
loss of antigens
When cutting sections for DNA or RNA extraction, all instruments and equipment must be pre-cleaned and wiped down with
RNAse
type of water used for floating sections for RNA extraction.
molecular grade water
Hardening of the celloidin block may be hastened by placing
chloroform
To avoid dehydration and shrinkage in section cutting, what should be done?
lubricated with 60-70% ethanol
essential for methods that require exposure of sections to acids and alkalis (especially ammoniacal silver solutions) during staining
adhesives
One disadvantage of using albumin
retention of stain which causes dirty background
