FINALS - MICROTOME, SECTION CUTTING, ADHESIVES, MOUNTING Flashcards
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
is 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
3 essential parts of microtome
block holder
knife carrier and knife
pawl, ratchet feed wheel and adjustment screws
microtome essential part where the tissue is held in position
block holder
microtome essential part where its primary purpose is for actual cutting of tissue sections
knife carrier and knife
microtome essential part where its purpose is 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
the wheel that is for forward and backward movement of the tissue block
course wheel
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.
what are the kinds of microtome
rocking
rotary
sliding
freezing
cryostat or cold microtome
ultrathin
this kind of microtome is for cutting serial sections of large blocks of paraffin embedded tissues
rocking microtome
this kind of microtome is for cutting paraffin embedded sections
rotary microtome
this kind of microtome is for cutting celloidin embedded sections
sliding microtome
this kind of microtome is for cutting unembedded frozen sections
freezing microtome
microtome for large blocks such as brain, whole organ
rocking microtome
this kind of microtome is for cutting sections for electron microscopy
ultrathin microtome
rocking (cambridge) microtome is invented by whom
paldwell trefall in 1881
is the simplest among the different types of microtomes
rocking (cambridge) microtome
rocking microtome is also called as
cambridge microtome
rocking (cambridge) microtome is consists of a
heavy base
2 arms (the lower arm 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
The Cambridge rocking microtome, available in how many sizes
two
has been used to
cut small and large blocks of paraffin tissues
cambridge rocking microtome
ROCKING (CAMBRIDGE) MICROTOME
a section is thereby cut as the tissue passes to the knife edge in what plane and thickness
slightly curved plane
10-12 u in thickness
rocking microtome where in the block
holder are brought together by __ motions
upward and vertical motions
rotary microtome is invented by whom
Minot 1885-86
rotary microtome is also called as q
minot microtome
used to cut paraffin embedded tissue, and is currently the most type used for both routine and research laboratories, especially for sectioning paraffin-embedded tissues
rotary (minot microtome )
a variant of rotary (minot microtome ) that is now available and can be ideally used to produced ribbons for serial sections
electrically driven rotary (minot microtome)
rotary or minot microtome can cut the tissue in what thickness
2-3 u but 4-6 is acceptable according to the nature of the tissue
based on the book, it’s between 3-5 um
this type of microtome where in the
knife is fixed in a HORIZONTAL position
rotary microtome
in rotary microtome, a heavier knife is used so there is less ___
vibration
in rotary microtome, The cutting angle (tilt) of knife
is adjustable, so it can ___tissue.
cut harder
rotary microtome
It can cut celloidin-embedded sections
by using a special holder to set the knife __.
obliquely
2 types of sliding microtome
standard sliding microtome
base sledge microtome
sliding microtome is used for ___ embedded tissues
celloidin embedded tissue
sliding microtome is developed by
adams 1789
this type of sliding microtome consist of two movable pillars holding the adjustable knife clamps allowing the knife to bet set at an angle for cutting celloidin sections
base-sledge microtome
this type of sliding microtome that is favored in laboratories where very hard tissue or large blocks are usually section
base-sledge microtome
such a machine is 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
the chuck of block holder is set on a heavy metal base which can be moved backwards and forwards under the knife
sliding microtome - base sledge
this sliding microtome, where the block remains stationary while the knife is moved backward and forward during the process of sectioning
standard sliding microtome
it was originally designed for cutting sections of very large blocks(whole brain )
base-sledge microtome
how long is the knife for base sledge microtome
24 cm
the angle of the knife used in base sledge is adjustable is and quite very long, 24 cm, that is why it requires less __
honing
ideal for resin-embedded decalcified bone
modern model of heavy duty base sledge microtome
a type of sliding microtome where the block remains stationary
while the knife is moved backward and forward during the process of sectioning
standard sliding microtome
type of sliding microtome
It was developed mainly for cutting celloidin embedded tissue blocks and is
inherently more dangerous because of the movable knife, which makes it
difficult to attach knife guards
standard sliding microtome
in both of these machines, the 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
it is the most dangerous type of microtome due to the movable exposed knife
standard sliding microtome
in manipulating standard sliding microtome, _____ motion is therefore required to manipulate the instrument.
a slow but very steady motion
freezing microtome is invented by
Queckett in 1848
describe the set up of freezing microtome
the stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe through which carbon dioxide passes from a cylinder
a simple level operated valve allows the release of rapid, intermittent bursts of carbon dioxide which will freeze the block holder and the tissue evenly
this process is for what type of microtome
freezing microtome
used to cut undehydrated thin to semi thin sections of fresh, frozen tissues
freezing microtome
freezing microtome is used in what instances
when rapid diagnosis is required
when histological demonstration of fat is needed
when certain neurological structures are to be studies
when sensitive tissue constituents to be studies are damaged or destroyed by heaty
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 freezing microtome is equipped with a stage upon which tissue can be quickly frozen using either ____
liquid carbon dioxide from a cylinder
or
low temperature recirculating coolant
the cutting action of the freezing microtome differs from those described previously because
the knife is moved whilst the tissue block remains static, same as sliding microtome
freezing, cryostat, and frozen microtome has same principle, but they differs in
freezing agent
is a refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning
cryostat
cryostat is also called as
cold microtome
cryostat consist of what type of microtome found inside the cold chamber
rotary microtome
what is the maintaining temperature of cryostat
-5C to -30C
average is -20*C
cryostat has a maintaining temperature of -5C to -30C (ave *-20C), adjustable thermostat, and is capable of freezing fresh tissues within how many minutes
2-3 minutes
cryostat can cut tissues of what size with EASE
4 u - four micra
cryostat provides a means of preparing thin sections of fresh frozen tissues especially for what techniques or studies
for fluorescent antibody staining techniques
histochemical studies
it is most commonly used for RAPID PREPARATION of urgent tissue biopsies for NTRAOPERATIVE diagnoses
cryostat
it is often housed in the frozen section room close to the operating room to allow direct consultation between surgeon and pathologist
cryostat
sections and usually transferred directly from the microtome knife to a slide or cover glass, all of which are maintained at a low temperature
cryostat
an ultrathin microtome is equipped with a ___ knife
glass or gem grade diamond knife
ultrathin microscope, using glass or gem diamond cutter is used to cut very thin sections which typically has what size
60-100 nanometer
the most common freezing agent is
liquid nitrogen with -190*C temperature
Ultrathin microtome is usually used for cutting ___ biopsies
renal biopsies
ultrathin microtome is usually for tissue embedded with __
epoxy resin
what type of microscope is the ultrathin microtome associated into
TEM - transmission electron microscope
what are done with microtome knife
trimming and section-cutting
3 basic types or shapes of microtome knives
plane-concave knife
biconcave knife
plane-wedge knife
plane-concave knife is usually in ___ mm length
25 mm in length
describe the sides of plane concave knife
one side of the knife is flat while the other is concave
less concave side of the plane-concave knife is for __
cutting celloidin-embedded tissue blocks on a sliding microtome
more concave side of the plane-concave knife is for __
paraffin sections on base-sled, rotary, rocking microtome
what type of microtome is the less concave side of plane-concave knife is recommended
sliding microtome
what type of microtome is the more concave side of plane-concave knife is recommended
base sledge microtome
rotary microtome
rocking microtome
the biconcave knife has approx ___ mm in length
120 mm
describe the knife of a biconcave knife
both sides are concave
biconcave knife is recommended for what microtome
rotary microtome - paraffin embedded sections
the plane wedge knife has approx ___ mm in length
100 mm in length
describe the knife of plane-wedge knife
both sides are straight
plane-wedge knife is recommended for what type of samples
frozen samples or for cutting extremely hard and touch specimens embedded in paraffin blocks, using a base sledge type of sliding microtome
the angle formed between the cutting edges
bevel angle
the bevel angle is normally about
27* to 32*
types of knives in electron microscope
glass and diamond knives
a type of knife that is used for trimming and semi-thin sectioning of tissue block
glass knives
a type of knife that is used to cut any type of resin block
diamond knives
a type of knife that is brittle and expensive, but very durable
diamond knives
why are plane wedge and plane concave knives are provided with backs?
to maintain the correct bevel angle throughout the honing
a good cutting edge should be made of good quality steel, a too soft cutting edge are likely to become
dull easily
a good cutting edge should be made of good quality steel, a too hard cutting edges are likely to produce
nicks or jagged edges and irregularities on the knife edge, thereby producing tears or striation on the tissue sections during cutting
a good cutting edge must be able to cut good sections from a paraffin wax block of about ____ microns thick without any serration noted on examination
2-3 microns
when do we use safety razor blades
safety razor blades may be used for patially calcified materials, paraffin sections and frozen sections
why do we need to hone and strop nicked and blunted ends of our knives?
to ensure optimum sectioning of tissue blocks
prevent gross irregularities on tissue sections
a process of removal of gross nicks on the knife edge to remove blemishes and grinding the cutting edge of the knife on the stone to acquire an even edge
honing
explain what’s coarse honing in honing process
removal of gross nicks on the knife edge to remove blemishes
explain what’s honing proper in honing process
grinding the cutting edge of the knife on a stone to acquire an even edge
relationship of sharpness and abrasive used in sharpening
the degree of sharpness is proportional to the fineness of the abrasive used in sharpening
a natural sharpening stone or hard grinding surface which serves to remove nicks and irregularities on the knife edges
carborundum
types of hones
belgium yellow
arkansas
fine carborundum
this hone is used for MANUAL SHARPENING when cutting edge has been rendered blunt or nicked
belgium yellow
this type of hone usually gives the best result
Belgium yellow
this type of hone gives more polishing effect than the Belgium yellow
arkansas
this hone is much COARSER than any other hones, and is used for badly nicked knives followed by either one of the other hones
fine carborundum
agent we can use to remove the scatted small particles of stones and metal during honing
soft cloth moistened with xylene
thin films we can use to cover the knife after honing for lubrication
mineral oil
clove oil
xylene
Liquid paraffin
soapy water
direction of honing
heel to toe direction
or
handle end to head portion
or
edge first
is the process whereby the “burr” formed during honing is removed and the cutting edge of the knife is polished
stropping
the purpose is to polish and sharpen the cutting edge,
stropping
is a process whereby tissues are cut into uniformly thin slices or sections with the air of a microtome
sectioning
3 general types of tissue sections
paraffin sections
celloidin sections
frozen sections
tissue section for paraffin-embedded tissue blocks, which may be cut by rocking and rotary microtome
paraffin sections
tissue section for celloidin embedded tissues which are usually cut by means of the sliding microtome
celloidin sections
tissue section for which may be cut from tissues that have been fixed and frozen with CO2 or fresh or fixed tissues frozen with the cryostat
frozen sections
factors to consider in sectioning
cutting depend upon the type of the tissue
the size of the block
the model or type of the microtome
— sections usually 4-6 microns in thickness for routine histologic procedure
— the knife is usually tilted 0-15* angulation on a microtome
a section is selected for staining and picked up onto a clean slide in vertical position
this process is called as
floating out
slide is immersed in the water bath in a near vertical position as close as possible to the section
this process is called as
floating out
temperature of the water bath during floating out
45-50*C
approx 6-10*C lower than the melting point of the wax used for embedding the tissue
substances that can be smeared onto the slides so that the sections stick well to the slides
adhesives
choice of slide and adhesive will be influenced by the ___ to be subsequently applied
staining methods
adhesives is not necessary for _____
routine staining, provided that the slides are clean and free from grease
applying adhesives is essential for methods that require exposure of sections to __
acids and alkalis (especially ammoniacal silver solutions during staining)
after adhesive and fishing out, we can dry the slides through ___
either leaving the slides in a 37*C incubator overnight
in wax oven at 56C - 60C for 2 hrs
dry the slides on a hot plate at 45C - 55C for 30 mns to 45 mns
a lower temperature and longer drying time is needed in what type of specimen
for more delicate tissues like the CNS tissue or brain
37*C for 24 hrs is needed
why do a lower temperature and longer drying time is needed for a delicate type of tissue
to avoid splitting and cracking of the section due to excess heat
instances when sections may float from the slide and adhesives 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 temperature
most commonly used adhesive because it is very easy to make, convenient and is relatively inexpensive
mayer’s egg albumin
advantage of mayer’s egg albumin
easy to make
convenient
relatively inexpensive
formula for mayer’s egg albumin
egg white 50 cc
glycerin 50 cc
common adhesives used
mayer’s egg albumin
dried albumin
gelatin
starch paste
plasma
mayer’s egg albumin composition
glycerin
egg white
thymol crystals
dried albumin composition
sodium chloride
dry albumin
thymol crystals
gelatin adhesive composition
gelatin
glycerol
phenol crystals dissolved in distilled water
plasma adhesive composition
plasma itself - readily available from outdated blood stored in blood banks
act as preservatives in mayer’s egg albumin and dry albumin
thymol crystals
starch paste as adhesive composition
powdered starch, 2 drops of hydrochloric acid, thymol crystals
adhesive usually used for transportation
gelatin-formaldehyde mixture
gelatin-formaldehyde component
1% gelatin
2% formaldehyde
coat the slides and dry at 37*C for 1 hr or overnight
adhesive widely used as a section adhesive in immunohistochemistry
Poly - L -Lysine
poly-L-Lysine is an aqueous detergent that can be purchased as a ___ solution
0.1% which is diluted with DW 1:10
adhesive useful in cytology, particularly for cytospin preparations of proteinaceous or bloody material
APES
3-aminopropylthriethoxysilane
last step in tissue processing that results in permanent histoloigical preparation suitable for microscopy
mounting
a syrupy fluid applied between the section and the coverslip after staining, setting the section firmly preventing the movement of the coverslip
mounting medium
purposes of mounting
it protects the stained section from getting scratched,
to facilitate easy handling and storage of the slides
to prevent bleaching or deterioration due to oxidation, thereby preserving the slides for permanent keeping
governs the contrast between cellular detail and the background, and also the transparency of the observed sample against the bright field of the microscope
refractive index
the mounting media must always have a refractive index higher than the mounted sample to impart more transparency
true or false
true
characteristics of a good mounting medium
it should be colorless and transparent
it should freely miscible with xylene and toluene
it should not dry to a non-stick consistency and harden relatively quickly
it should be resistant to contamination (particularly microorganism growth)
it should not cause shrinkage and distortion of tissues
it should not leach out any stain or affect staining
it should not change in color or pH
it should be compatible with the adhesive in use
it should set without crystalling, cracking or shrinking (or otherwise deform the tissue being mounted) and not react with, leach or induce fading in stains and reaction products (including those from enzyme histochemical, hybridization, and immunohistochemical procedures)
as a general rule, a paper label bearing the ___
patient’s name, section number, and preferably the staining method used
is attached to the slide for proper identification, while also avoiding any damage to the sections caused by wiping the “wrong” side of the slide
2 types of mounting media
aqueous media
resinous media
are used for mounting sections from distilled water when the stains would be decolorized or removed by alcohol and xylene
aqueous mounting medium
aqueous mounting medium is made up of
gelatin,
glycerin jelly or gum arabic
glycerol,
sugar
and a preservative solution
which of the composition of aqueous mounting media is to solidify the medium
glycerin jelly or gum arabic
which of the composition of aqueous mounting media is for prevention of cracking and drying of the preparation
glycerol
which of the composition of aqueous mounting media is to increase refractive index
sugar
has a low refractive index, is moderately transparent and evaporates easily, hence is good only for temporary mounting
water
the refractory index of it improves the image quality and also supports the specimen
water
wet mounts cannot be stored over extended time periods as the water evaporates
t or f
truelalu
this mounting medium may also be used as a preservative
glycerin RI.1.46
has a high index of refraction and provides greater visibility if slightly diluted with water (for moist sections)
glycerin RI: 1.46
very suitable semi-permanent mounting medium
glycerin R.I: 1.46
miscible with water, is inexpensive, and is non poisonous
glycerin R.I: 1.46
disadavantages of glycerin R.I: 1.46
difficult to prepare slides that are truly permanent in nature
slowly run off a slide that is tilted
do not stack slides for long as the pressure will squeeze glycerin from the mounts
glycerin will eventually evaporates and air will penetrate under the coverslip
glycerin can be attacked by microorganism
commonly used to mount sections for IMMUNOFLUORESCENCE and glycerol may be added to other agents to retard drying and cracking
phosphate buffered glycerol RI: 1.47
glycerin jelly (kaiser’s 1880)’s preservative
phenol crystals
is the standard mounting medium used when dehydration and clearing with xylene cannot be made (as in fat stains)
glycerin jelly (kaiser 1880) RI: 1.47
preservative of farrant’s medium RI: 1.43
sodium merthiolate
does not solidify upon storage and therefore does not need to be heated before use
farrant’s medium RI: 1.43
takes a longer time to harden and may therefore require ringing
farrant’s medium RI: 1.43
farrant’s medium RI: 1.43
__ may be used as a substitute of sodium merthiolate for preservation of the medium
arsenic trioxide
APATHY’S MEDIUM (RI = 1.52)’s preservative
thymol crystals
used for methylene blue-stained nerve preparations and and as a general purpose aqueous mountant
APATHY’S MEDIUM (RI = 1.52)
why is APATHY’S MEDIUM (RI = 1.52) has the highest RI
due to the presence of PURE CANE SUGAR OR SUCROSE
one of the most useful aqueous mountants for fluorescent, being virtually non fluorescent
APATHY’S MEDIUM (RI = 1.52)
sets quite hard, has a higher RI, and does not require ringing
APATHY’S MEDIUM (RI = 1.52)
aqueous mounting media recommended for mounting frozen sections from water
BRUN’S FLUID
mounting media that are used for preparations that have been dehydrated and cleared in xylene or toluene, and are recommended for majority of staining metjods
resinous mounting media
resinous mounting media may be divided into 2, what are these
natural and synthetic resins
the most important synthetic resins are used for embedding __
undecalcified bones, and for electron microscopy
canada balsam came from canadian tree called
abus balsamea
this natural resinous mounting media can solidify but can be dissolved by xylene in an incubator at 37C or paraffin oven at 58C
Canada Balsam (RI = 1.524)
it is transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation
Canada Balsam (RI = 1.524)
what is the component of Canada Balsam (RI = 1.524) that maintain its neutral reaction
calcium carbonate chips
is recommended for whole mounts and for thick sections because it does not shrink much
Canada Balsam (RI = 1.524)
recommended for small tissue sections but not for whole
mounts because of shrinkage produced on drying
DPX - (Dibutyl Phthalate and
Xylene) (RI = 1.532)
colorless, neutral medium in which most standard stains
are well preserved.
DPX - (Dibutyl Phthalate and
Xylene) (RI = 1.532)
It tends to set quickly and, in doing so, often retract from
the edge of the coverslip.
DPX - (Dibutyl Phthalate and
Xylene) (RI = 1.532)
is a synthetic resin mixture in xylene, available in a
pale yellow or colorless solution.
XAM (RI = 1.52)
a resinous mounting medium that Dries quickly without retraction, and preserves stains
well
XAM (RI = 1.52)
synthetic resin which is soluble in xylene
* generally preferred over D.P.X
CLARITE (RI = 1.544)
other recommended synthetic mounting media
- Permount (made by Fisher Scientific)
- H.S.R. (Harleco Synthetic Resin)
- Clearmount (Gurr
Permount is made by
fisher scientific
H.S.R stands for
harleco synthetic resin
process of sealing the margins of the cover-slip to prevent
the escape of fluid or semi-fluid mounts and evaporation
of mountant, to fix the coverslip in place, and to prevent
sticking of the slides upon storage.
ringing
The term “ringing” originated because round coverslips
were initially used and the coating applied in the form of
a circle or “ring.”
t or false
true
kronig cement composition
2 parts paraffin wax mixed with 4-9 parts powdered colophonium resin
heated and filtered
this ringing coat that is a cellulose adhesive
durofix
