SECTION-CUTTING & FLOATING OUT

• MICROSCOPE SLIDES

• CHEMICAL RESISTANT PEN FOR LABELLING

• FORCEPS / WOODEN APPLICATOR STICK

• WIPES, MICROTOME BLADDES

• EMBEDDED TISSUE CASSETTES

• ICE WATER DISH/TRAY

• GAUZE

• CAMEL’S HAIRBRUSH

• SLIDE RACK

• ROTARY MICOTOMR

• FLOATATION (WATER) BATH

SUPPLIES & EQUIPMENT REQUIRED FOR

PARAFFIN SECTION CUTTING

(Knife holde) (block holder) (Feed mechanism) (parallel) (straight ribbon)

SECTIONING TECHNIQUE

Insert the knife in the ________& screw tightly Fix the block in the __________ and ensure it is secure. _____________ is adjusted until the wax block is almost touching the knife. Ensure that the whole surface of the block will move _________ to the knife so that ___________ of sections is obtained

(mucosa) (epidermis) (dense tissue)

APPROPRIATE ORIENTATION OF THE SPECIMEN IN THE TISSUE HOLDER

INTESTINE:

blade passes through the ____________ last

SKIN:

blade passes through the ____________ last

CERVIX

better to present a point of______________

to the bladder rather than a straight on

(block) (15-20um) (3-5um) (ice) (frictional heat)

SECTIONING TECHNIQUE

All screws should be tight to avoid faulty

sectioning

For _________ section thicknes of

around ________ with a rough knife is taken

Sharp knife is used for sectioning

Reset the thickness gauge to required thickness

_______ recommended for routine work

Keep the blocks on _____ to make the wax hard

which would have become soft by __________.

There should be a smooth continuous plastic

flow of the sections in the form of a ribbon

(FLOATATION/FLOTATION) (FORCEPS) (WATER BATH)

________/________ to straighten the tissue ribbon comes off it is held gently with a fine moistened brush or ______ and then transferred to ___________

(45-50C) (6-10 lower) (melting point)

section is then floated on water bath ________ or _________ than the (________) of the wax to avoid any chance of mix-up float out sections from one block at a time

(DURING SECTIONING) (SECTIONS FAIL TO FORM RIBBONS) (not parallel) (Re-trim) (re-orient) (too hard) (lower melting point) (tilted) (reduce) (too thick) (dull) (Hone and strap)

FAULTS/PROBLEMS

OBSERVED (_____________)

(_______________)

REASON	REMEDY
Surfaces and edges of the block is _________	___________ the block
Horizontal surface of the block is not parallel to the knife	Re-adjust and __________ the block

Paraffin wax is _________	Coat horizontal edges of the block with wax of __________
Knife is __________ too much	___________ the tilt
Sections are __________________	Readjust the thickness of the sections

Knife is __________	________

(ROLLS UP ON CUTTING) (BROKEN) (blunt) (Reduce) (dirty)

SECTIONS (_____________) SO THAT THEY

ADHERE AND GET (______) AGAINST THE KNIFE

EDGE

REASON	REMEDY
Knife is __________	Sharpen the knife
Tilt of knife is too great	____________ the tilt

Knife edge is ________	Clean the knife edge

(CURVED, CROOKED OR UNEVEN) (Heat) (dull) (uniformly sharp edge) (Re-trim) (block)

RIBBON IS (_______, _________, ________) INSTEAD OF STRAIGHT

REASON	REMEDY
________ or _______ on the knife, producing an irregular knife edge	Adjust the knife so that knife edge will present a __________________ to the block or sharpen
Edges of the block are not parallel but round or wedge shaped	_____________ the block

Knife is not parallel to the _____________	Readjust the knife and block

(COMPRESSED, WRINKLED OR JAMMED) (Re-sharpen) (warm & soft) (ice water) (too thin) (Readjust) (loose) (Reduce)

SECTIONS ARE (_______, ________, ________)

REASON	REMEDY
Knife is blunt or dull	___________ the knife
Paraffin block is ___________ and ____________	Cool the block on_________ until firm

Knife edge is coated with paraffin	Clean the knife edge
Sections are ______________	_____________the thickness of the section
Microtome set screw is _____________	Tighten the screw

Tilt of knife is too vertical	______ the tilt

SECTIONS ARE ____________

(width of the each section is less than that of the block)

REASON	REMEDY
Bevel of knife is lost due to incorect sharpening	Re-sharpen , using a knife back or automatic knife sharpener

(LOWER PART) (small) (compressed) (increase)

RESISTANCE IS FELT ON THE (________) OF THE

SECTION DURING CUTTING

REASON	REMEDY
Tilt of knife is too _________, paraffin block is therefore ___________ against the base of the knife towards the end of stroke	__________ the tilt

(bubble) (dirt) (Re-embedded) (under processed) (calcium) (decalcification) (bare sledge)

A HOLE IS FORMED IN THE SECTION

REASON	REMEDY
___________ or ____ formed in the embedding medium	____________ in freshly filtered wax if necessary
Tissue is not processed properly and will not form a section	Re-process tissue

________________ portion of tissue bursts on contact with warm water	Re-process tissue
Hard spot in tissue due to __________	Once embedded in paraffin wax, ________________ is impractical; use a ______________ microtome with a wedge knife

(UNEQUAL THICKNESS) (not cleared) (tighten) (smaller fragments) (hard)

SECTIONS OF (_____________) ARE PRODUCED

REASON	REMEDY
Tilt of knife is too great or bevel is ____________ , hence object is compressed against the knife edge	Reduce the tilt
Clamp set screw on knife or block holder is loose	___________ the screw

Blocks are too large	Cut blocks into _________
Blocks are ___________	Soften the blocks in detergent or phenol

(ADGERE TO THE KNIFE) (static electricity) (knife edge) (dull) (reduce)

SECTIONS (________________) OR OTHER PARTS OF THE MICROTOME

REASON	REMEDY
_____________ due to low atmospheric humidity	Breathe out or blow gently on the block and knife to break up static electricity, or boil water in the room to increase humidity
Knife edge is dirty	Clean the ___________

Knife edge is __________	Sharpen the knife
Knife tilt is too great	___________ the tilt

RIBBON IS SPLIT OR LENGTHWISE VERTICAL

SCRATCHES ARE SEEN ON SECTIONS

REASON	REMEDY
__________ or damage on the knife edge	Sharpen the knife
_________ embedding	__________ in freshly filtered wax

Knife edge is ____________	Clean knife edge with xylene
Tilt of knife is too great	Reduce the tilt

(soft) (warm)

SECTIONS ARE LIFTED FROM THE KNIFE ON UPSTROKES

REASON	REMEDY
knife tilt is too great	Reduce the tilt
knife is dull	Sharpen the knife

Paraffin is too _____ or room temperature is ______	Cool paraffin wax in ice water

(HORIZONTAL OR PARALLEL LINES OR FURROWS) (chatters) (hardness) (phenol)

(_________________________________)

ACROSS THE SECTIONS (________) ARE SEEN

REASON	REMEDY
Knife edge vibrates due to ___________ of tissue	Treat with _________ during processing or colloidionize
Tilt of knife is too great	Reduce the tilt

SECTION CUT IS SOMETIMES THIN, SOMETIMES THICK

REASON knife is blunt	REMEDY Sharpen the knife
knife is not __________ properly	Adjust the knife so that knife edge will present a uniformly shard edge to the block or sharpen
Knife or block holder is loose	Tighten adjusting and locking screws

Knife tilt is _____________ that block is _____________ by bevel and section is not cut	______________ the tilt

(HAND METALIC) (RINGING) (reduces) (too hard)

KNIFE MAKES A __________ SCRAPING OR (_________) SOUND ON BACKSTROKE, WHEN SECTION IS CUT

REASON	REMEDY
Tilt of knife is too slanted or too big	___________ the tilt
Tissue is ____________	Take fresh block treated with phenol during processing

knife blade is too thin	Change the knife

(crumbles) (not adequate)

FROZEN TISSUE _________ AND COMES OFF

THE BLOCK HOLDER WHEN CUT

REASON	REMEDY
Freezing is __________	Refreeze the tissue block

(Chips) (Frozen)

FROZEN TISSUE ________ INTO FRAGMENTS

WHEN CUT

REASON	REMEDY
Tissue is ____________ too much	Warm the tissue with the fingers

RIBBONS ARE (CROOKED)

REASON	REMEDY
Top and bottom edges of block are not parallel to edge of blade / sides of block are not perpendicular to the blade	Adjust the block holder to make the block edges parallel to the knife

SECTIONS ARE TOO THICK

REASON	REMEDY
Wrong __________ setting	Microtome needs _______________

(OPAQUE) (ALCOHOL) (clearing) (clearing) (12 hours)

TISSUE IS _________, SECTION CUTTING IS

DIFFICULT DUE TO PRESENCE OF (____________)

REASON	REMEDY
Insufficient ___________	Repeat (_________); if object has already been embedded, prolong clearing up to _____________, then re-embed

(SHRINKS AWAY) (dehydration)

TISSUE (____________) FROM WAX WHEN TRIMMED

REASON	REMEDY
Insufficient _____________ , therefore incomplete clearing and impregnation	Repeat the whole procedure

(Crystalline) (not cooled)

ON TRIMMING, WAX APPEARS __________

REASON	REMEDY
Contaminated wax	Re-embed in freshly filtered wax
Block ___________ rapidly enough	Re-embed in freshly filtered wax

(MOIST AND CRUMBLES) Insuffucent

PARAFFIN BLOCK, AFTER COOLING IS (________&__________)

REASON	REMEDY
________________ paraffin	Repeat paraffin impregnation, then re-embed

(ADHESION) (washing out) (Adhesive) (stick) 1.Mayer’s Egg Albumin 2.Dried Albumin 3.1% Gelatin 4.Gelatin-formaldehyde mixture 5.Poly-L-lysine 6.APES/APTES (3-aminoprophylthriethoxysilane)

(___________)

performed to prevent

_________ of tissue sections

during staining

(_________) is a substance which

can be smeared onto the

slides so that sections ______

well to the slides

(Mayer’s Egg Albumin) (egg white + glycerin + thymol)

ADHESION

(_____________)

(______+______+_______)

• most commonly

used

(Dried Albumin) (dried albumin + NaCl + thymol)

(______________)

(____+_____+_____)

sections are

dried and

stored in 70%

alcohol until it

is ready for

staining

(1% Gelatin) (gelatin + glycerol + phenol)

(__________)

(_____+_____+______)

added to the

waterbath during

flotation rather

than applying it

on the slides

Gelatin-formaldehyde

mixture (Gelatin + formaldehyde) (37C)

(____________)

(_______+__________)

slides are coated and allowed to dry at ______ for one hour or overnight prior to use

(Poly-L-lysine) (0.01%) (immunohistochemistry)

(___________)

aqueous detergent

diluted to ________

concentration widely

used in

(___________)

(APES/APTES)

(3-aminoprophylthriethoxysilane) (cytology)

(__________)

(___________________)

very useful in _______,

particularly for cytospin

preparations of

proteinaceuos for

bloody material

best action adhesive

available and coated

slides can be stored for a

long time

(Fishing out) (10 C lower (6-10 lower) (ORIENTATION)

FISHING OUT, ORIENTATION,

DEPARAFFINIZATION & DRYING OUT

___________: water bath should have a temperature that is _________than the melting point of the wax.

(________________): correct positioning of the tissue section/ribbon on the slide

(DEPARAFFINIZATION & DRYING SECTIONS) (56-60 C) (30 - 45 mins) (37 C) (Blower type)

(____________________________________)

• Wax Oven (_______ for 2 hours)

• Incubators (overnight)

• Hot plate (45°C-55°C for _______)

• Alcohol lamp/ Bunsen Flame

• Delicate tissues: ____for at least 24 hours

• _________ electric slide dryer (50-55°C for 20-30 mins)

(POST-MORDANTING) (secondary fixation) (K2CrO4) (mordant & secondary) (5-10 minutes) (HgCl2) (Picric Acid) (preservation of lipids) (mitochondria)

(______________________)

• _____________ (post-chroming)

• 2.5-3% _______ for 24 hours

• used primarily as (________&__________) as fixative

• (_______) in either

• -Aqueous solution of (_____)

• -Aqueous solution of (___________)

• Purpose : to improve the ___________ and

• lipoprotein materials that would otherwise be

• removed by processing reagents

• improve the preservation of ___________ and

- myelin