i) rotary microtome
-for production of large
number of sections
-serial section
ii) floatation bath
-thermostically controlled
-for paraffin wax 56oC
melting point water bath temperature is about
45oC
-a variety of fluids are
recommended from distilled water to quite high
concentration of alcohol
iii) hot plate or drying oven
-recommended drying of
section onto slides should take place at
temperature as low as 37oC
-section dried at low temperature
drying is prolonged - over night
-drying section on a hot plate is necessary if
staining is to be carried out
urgently
iv) brush, seeker, forceps
-necessary during sectioning for removal of folds
v) slides,
glass marker
- size 76mm x 25mm, 1 to 2mm thick
-mark slides with a glass marker
vi) section adhesives
-most tissue section do not
requires any adhesive provided sections
are
initially dried adequately on to slides
-if using adhesive solution tend
to remove section from slides so
adhesive should be use
-types of adhesive
-albumen
-gelatine
-starch
-cellulose
-sodium silicate
-resin
-tissues
impregnated with ester wax or polyester wax will usually require the use of
section adhesive - to minimize loss
a) Mayer`s glycerol albumen
Fresh egg white 50cm3
Glycerol 50cm3
Sodium salicylate 1cm3
-mix and agitate ingredient
-filter through coarse filter
paper
-thin smear is needed on the
slide
Sectioning
i) setting up the microtome
-angle of slope of knife at 90o (clearance 5-10 o )
-tighten knife clamp screws securely
block clamp screw must firmly
-exposed ends of knife must be protected
ii) trimming of tissue block
-to trim any surplus wax
-to expose suitable area of tissue for
sectioning - thickness at 20um
-on exposing - section thickness
set to 0.5-4um
-top and bottom of block parallel and
horizontal to the edge of knife at the
moment of impact
iii) cutting sections
-all tissues desired on the slide should be
exposed on the surface
-no
scratch marks on the surface
-if scratch marks are visible the knife
must be moved laterally
the
face trimmed again
-speed of block is important
-softer
tissues - at a slower rate
-optimal speed is obtained through
experience
-maintain
a regular cutting rhythm
-a ribbon section is produced
-use fine forceps to hold the free end of the
ribbon
-use soft paint brush to brush away the last
section from the knife
-transfer ribbon to water bath
-to obtain flat section its is necessary
to spend time in the cutting and gentle
stretching of the ribbon - before floating on
the water surface
iv) floating out sections
-action in floating out must be smooth with
trailing end of the ribbon
making contact with the water
-slight drag when ribbon touches the water will
produce tension in ribbon
remove folds from section
-when
ribbon has come to rest on the water - remaining wrinkles and folds
are removed by teasing a part using forceps or
seeker
-prolonged floating out of section on the
water bath must be avoided as
tissues may expand and become distorted
-advantage if water bath has a black base -
section are more easily seen
-some
tissues eg. cartilage are difficult to
flatten on the water surface
- to overcome this prolonged
floating on the water bath - teasing with forceps
or seekers
-trimming away surplus wax from
the block
v) picking up sections
-is done by immersing the slide
-vertically in the water bath
-position the section into contact with
the slide
-drain it in a vertical
position for several minutes
vi) drying sections
-if
staining is required urgently drying section on a hot plate is necessary
-drying
time is 10 minutes
-dried in an oven - 45 minutes at 45o C
- section loss if less than 30
minutes
-advantage of oven drying - dust is less
likely to settle on sections
-after removal from hot plate or oven - cooled
sections are stored in dust
free containers
-if overnight the temperature is 37o
C
Faults
and remedies in paraffin wax sectioning
i) Sections will not join to form a
ribbon
-Cause -wax
too hard
-dust on knife edge
-knife angle too wide or narrow
-Remedy -warm or re-embed
-clean with xylene
-adjust angle
ii) Sections roll into a coil
-Cause -knife blunt
-knife angle too wide
-section thickness too great
-Remedy -replace knife
-reduce knife tilt
-reduce section thickness
iii) When sections are curved
-Cause -knife blunt in one area
-excess wax in one side
-Remedy -use different part of knife or replace
with a new one
-trim away excess wax
iv) Splitting of sections
-Cause -damage in knife edge
-hard
particles in tissue
-hard
particles in wax
-Remedy -use different part of knife or replace
with a new one
-remove
particles from tissue
-re-embed
in fresh wax
v) Compression of sections
-Cause -blunt knife
-angle knife too wide
-wax too soft
-Remedy -replace with a new knife
-reduce knife tilt
-cool block in refrigerator
vi) Areas of tissue blocks not present in
sections
-Cause -incomplete impregnation of tissue
-wax block become detached
-Remedy -
return tissue to vacuum impregnation container for a few
hours
-re-attach
block
vii) Sections expand and disintegrate on water
surface
-Cause -poor impregnation of tissues
-water temperature too high
-floatation bath dirty
-Remedy -return
tissue to vacuum impregnation container for a few
hours
-cool water
-clean floatation bath
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