SECTIONING Flashcards by FRANCHESCA MAUREEN CUISON

Usual length: 25mm

Profile A: Plano concave

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Used on sliding, rotary and
rocking microtomes.

A, PLANO

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Extremely sharp, but are
also very delicate and are
therefore only used with
very soft samples

A, PLANO

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Length 100-250mm (usual
length: 120mm)

B, BICONCAVE

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Introduced by Heifflor

● Classify knife shape with
concavity on both sides.
● Less rigid

B, BICONCAVE

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Prone to vibration.

Recommended for cutting
paraffin-embedded
sections on a rotary
microtome.

B, BICONCAVE

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More stable and find use in moderately hard materials,
such as in epoxy or
cryogenic sample cutting.

C, WEDGE PROFILE

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Used in all types of
microtomes to cut all types of
tissues.

C, WEDGE

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Both cutting surfaces are
plane or flat.

● Known as the standard knife
profile.

● Most commonly utilized steel
knife for routine
histopathology.

● More rigid knife than Profiles
A and B.

C, WEDGE

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Used for sectioning FFPE
tissues.

C, WEDGE

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Size: 100 – 350 mm.

C, WEDGE

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This profile cannot be ground
as sharp as Profiles A and B.

C, WEDGE

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With its blunt edge, raises
the stability of the knife,
while requiring significantly
more force to achieve the
cut.

D, CHISEL PROFILE

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Caused by defect in the
blade edge, calcium,
bone, or hard material in
the specimen

VERTICAL SCRATCHES

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Ensure at the beginning
of the sectioning that the
block holder is adjusted
so that the block face
and the blade are
perfectly parallel.

TRUE

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Occur when block is
faced too aggressively.
● The specimen is either
dehydrated or
improperly processed.

HOLES IN THE SECTION

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Ensure to chill the block
with ice before cutting
and discard ribbons until
the hole disappear.

Facing the block less
aggressively, with
smaller micrometer
advances of the block for
each section removed.

HOLES IN THE SECTION

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Commonly occurs in
very hard tissue such
as uterus or in over
fixed tissue.

It is the macroscopic
type of chatter
commonly caused by
loose clamping of blade
or block.

WASHBOARDING OR UNDULATION

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Proper clamping of blade
and block.

Ensure the block holder
shafts is not over
extended.

Ensure the microtome is
in good working order.

Decrease the blade tilt.

TRUE

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Occur when the block
holder is not parallel to
the blade.

● One side of the block is
exhausted while
attempting to get a
complete section of the
block face.

BLOCK FACE UNEVENLY SECTIONED

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● Ensure at the beginning
of the sectioning that the
block holder is adjusted
so that the block face
and the blade are
perfectly parallel.

True

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Result when the
horizontal edges (to and
bottom) of the block are
not parallel.

If the lower block edge
is not parallel to the
knife edge.

COOKED RIBBONS

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The upper and lower
edge should be parallel.

The lower block edge is
parallel to the knife edge.

No problem in the blade
edge.

TRUE

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Commonly caused by
dull blade.

Could result from too
hard paraffin, too much
blade tilt

FAILURE OF RIBBON TO FORM

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Background staining may be detected due to its uptake of dyes. Preservative is added to prevent putrefaction (decomposition of proteins). Glycerol is also added to increase viscosity and prevent complete drying.

ALBUMIN

More effective if drying of sections takes place above the coagulation point of the albumin MOST COMMON

ALBUMIN

Equal parts of glycerin, distilled water, and egg white are mixed filtered through coarse filter paper. Thymol crystal is added to inhibit the growth of molds, solution kept in refrigerator. Small quantity of the solution is smeared over the surface of the slide immediately before mounting sections from the water bath.

ALBUMIN (MEYERS EGG)

Provides firmer attachment than albumin. Has to be gently heated before use to melt the gelatin. Shouldn’t be kept molten for long periods as in will lose its ability to solidify. Prepared as a 5% stock solution, aliquoted in amounts needed to produce a final concentration of 0.01% in the water bath(2 mL per liter), then refrigerated. Add one aliquot to the water in a tissue flotation bath and stir to distribute

GELATIN USP

Greater adhesion than gelatin

STARCH

Disadvantage: ○ Stains with many dyes.

STRACH

In the form of 1% Methyl cellulose Advantage: Not staining to any appreciable extent with commonly used in stains of histochemical reagents.

CELLULOSE

Use as a general-purpose section adhesive. ● No production of background stainining

POLY-L-LYSINE

Advantages: ○ Little tendency to staining with most dyes; not affected by the use of mild alkaline solutions

SODIUM SILICATE

Commercial syrup = 1:10 dilution ● Has strong adhesive properties

SODIUM SILICATE

Disadvantages: ○ Blackening in some silver impregnation techniques, in some reticulin methods, and red staining in methyl green pyronin technique.

SODIUM SILICATE

Greatest adhesion.

RESINS

Diluted 1:10 with acetone ● Little affect by most fluids in any treatment of sections

RESINS

A new advance in section adhesion. ● Withstand repeated washings with a variety of inorganic solvents.

APES

used as an adhesive for enhancing chromosome spreading on glass slides and for in situ hybridization of frozen sections

APES

Produces improved section bonding over other commonly used adhesives such as poly-L-lysine, glycerin , albumin, and gelatin and withstands protease digestion. There is no background staining.

APES

Does not seem to interfere with the routine histological or immunostaining methods. The other way to make a positively charged glass surface is to treat it with a solution of 3-aminopropyltriethoxysilane(APES) in the presence of catalytic traces of water. The 0.1% or so of water ordinarily present in acetone is enough.

APES

The positive charge of APES-treated (silanized) slides is like that imparted by Poly-L-lysine, but covalent bonding to the glass ensures that it cannot be washed away:

TRUE

Usually cut between 10 – 15 μ in thickness.

CELLIODIN

Block is trimmed the same way as paraffin blocks, leaving 3-5 mm of celloidin all around the tissue but do not require hardening by chilling before cutting.

CELLOIDIN

To avoid dehydration and shrinkage, sections are usually cut by the Wet method– both sections and block are kept moist w/ 70% alcohol during cutting.

CELLIODIN

Celloidin sections do not come off in ribbons and have to be collected into 70% alcohol immediately. Stored in jars w/ 70% alcohol in tightly fitting lids and finally mounted on to slides after they have stained.

TRUE

Used to flatten the sections and prepare them for mounting onto slides.

FLOATATION WATER BATH

Used to flatten the sections and prepare them for mounting onto slides.

FLOATATION WATER BATH

Wax is removed with a sharp knife (cutter knife/kitchen knife) until about _____ remains on all sides of the tissue

block trimming - 4-60mm

Done at approximately 30 mm at a time until the tissue surface is exposed.

COURSE TRIMMING - 30mm

Set thickness adjuster at 15 mm or advancing the block using the coarse feed mechanism; surface block is trimmed away until the entire tissue surface has been partly exposed

FINE TRIMMING - 15mm

Set thickness adjuster at 15 mm or advancing the block using the coarse feed mechanism; surface block is trimmed away until the entire tissue surface has been partly exposed

FINE - 15mm

Rocking and rotary microtomes

PARAFFIN

Rocking and rotary microtomes

PARAFFIN

Usually by means of a sliding microtome.

CELLOIDIN

Is the straight line formed by intersection of 2 planes, the cutting facets(Length = 0.1-0.6mm).

cutting edges

Angle between the planes. ○ Angle formed between the cutting edge (planes) of the microtome knife.

BEVEL ANGLE (27-32)

Angle between the sides of the knives.

WEDGE ANGLE (15)

Angle between cutting facet and block of tissue. ○ Angle formed by a line drawn along the block surface and the lower bevel of the knife.

clearance angle (5-10)

Angle between upper surface of the cutting facet and the surface of the block. ○ Angle between the upper bevel of the knife and a line at 90° to the block surface.

rake angle

Facet angle other names:

bevel and cutting

also added to increase viscosity and prevent complete drying.

GLYCEROL

is added to inhibit the growth of molds, solution kept in refrigerator

THYMOL CRYSTALS

This stain is used to demonstrate mucin/mucus in tissue sections?

alcian blue

Metachromatic staining is best demonstrated in which of the following?

formalin fixed

Which of the following stains black in Jones Methenamine Silver?

TYPE IV COLLAGEN

You saw black fibers on a yellow background in the tissue section. Which of the following stains might have been used?

fontana massons

Keratin stains in the standard trichrome staining.

red