SECTIONING 徐智慧/ TYPES OF HONES

Sectioning : cutting tissue uniformly into a thin slice/section with the aid of a Belgium yellow/ hone ▪ for manual sharpening
machine to facilitate the studies under the microscope. ▪ usually gives best result
Microtome : a machine/instrument designed for actual cutting of thin section. Arkansas ▪ gives more polishing effect
Fine carborundum ▪ much coarser
KINDS OF MICROTOME
▪ used for badly nicked knives
Sliding Base-sledge type
(celloidin- - 2 movable pillars holding the knife Plate glass hone ▪ use diamanthine for final polishing
embedded - chuck/block holder is set on a heavy metal base Machine hone ▪ glass disc or wheel driven by electric motor
section) - block holder is raised towards the knife
Std. sliding type COMMON LUBRICANT USED FOR HONING
- The block remains stationary - mineral oil
- the knife is moved backward and forward during sectioning - clove oil
Rotary - For paraffin-embedded sections - xylene
- most common type used for routine & research laboratories - liquid paraffin
Freezing - For unembedded tissue - soapy water
- a simple lever operated valve release a rapid, intermittent burst
of CO2 to freeze the block holder CARE AND USE OF MICRTOME KNIVES
▪ For rapid Dx ▪ perfect edge: junction of the smooth plane surface 14°
▪ For histological demo of fat ▪ use fine yellow Belgian water stone to remove large nicks
▪ For the study of neurological structure ▪ size 8’’ x 3’’
▪ For the study of sensitive tissue constituent which are ▪ keep the knife flat when being honed
damaged/ destroyed by heat ▪ finish the edge on a leather or linen strop
Rocking - For serial sections of large blocks of paraffin-embedded tissue ▪ wipe the knife clean with soft cloth before and after stropping strokes
- produce 60-90sections with ease ▪ use gentle pressure when stropping
▪ Lower arm : resting on pivots and supporting column ▪ avoid speed in stropping
▪ Upper arm : carry the block holder on one end by a screw ▪ leather strop require oiling before use. Use castor oil or vege oil to treat the
Ultra thin - For electron microscopy strop applied at the back of the strop (not on the surface)
sectioning ▪ mineral oil destroys the leather strop, done allow to come in contact with the
microtome strop
▪ wax shouldn’t come in contact with the strop
MAJOR PARTS OF A MICROTOME
Block Holder/ chuck Holds the tissue block in place TYPES OF TISSUE SECTIONING
Knife carrier and knife Actual cutting of tissues PARAFFIN SECTIONS
Pawl, ratchet feed Line up the tissue in proper position to the knife, ▪ Prep: trim the block until all sides are parallel
wheel, adjs.screw Adjust proper thickness of the tissue for successive
▪ Adhesive: for entailing significant exposure of section to acids and alkalis, but
sections
cannot be used for protein histochemical investigation
Mayer’s albumin Egg white + glycerin + thymol crystal
CARE OF THE ROTARY MICROTOME
▪ after cutting, brush away with soft brush: all accumulated paraffin and tissue Dried albumin Dried albumin + NaCl + thymol crystal
Gelatin Gelatin + water + glycerol +phenol crystal
▪ wipe clean all metal parts with xylol
Gelatin chrome Can be added to water bath.
▪ avoid continuous application of xylol to the rest of the machine (can remove
alum
the painted finish)
Starch paste Powdered starch + HCl + thymol crystal
▪ Dry the machine carefully especially the knife holder Plasma Outdated blood stored in blood banks
▪ keep the machine well oiled to prevent rust formation
▪ Actual Sectioning
▪ keep the moving parts of microtome oiled -nervous tissue, lymph nodes : slow, gentle motion
▪ cover the microtome when not in use (prevent accumulation of dust and dirt) -the harder the tissue, the harder & cooler the block should be
(may interfere the sectioning) ▪ Floating out bath
45-50C or 6-10C lower than the melting point of paraffin
MICROTOME KNIVES ▪ Drying of sections
PARTS OF MICROTOME KNIFE -wax oven 56-60C (2hours)
Cutting edge Cutting facet, made of good quality steel. -incubator 37C (overnight)
Able to cut good section from a paraffin wax block without -hot plate 45-55C (30-45 min)
any serration note on examination. -blower type electric slide dryer 50-55C (20-30min)
Wedge -bunsen flame until the wax melts
Knife back Spring-loaded semicircular sheet of metal slipped on the knife -nervous tissue: incubation overnight 37C
Bevel angle Angle between the cutting edge: 27-32
Wedge angle Angle formed by the sides of the wedge knife (5-14) DIFFICULTIES ENCOUTERED IN SECTION CUTTING
Clearance angle Angle between cutting facet (5-15) FAULTS REASON REMEDY
▪ prolonged fixation Soften tissue by soaking oil in a
KINDS OF MICROTOME KNIVES ▪ prolonged dehydration smaller dish or bowl (w/water)
with detergent, phenol or
Plane concave 250mm, 1 side is flat, 1 side is concave ▪ prolonged clearing
molliflex
Biconcave 120mm, both sides concave Brittle/hard tissue ▪ prolonged paraffin
Plane wedge 100mm, both sides straight infiltration in over heated
paraffin oven
CARE OF MICROTOME KNIFE ▪ drying out of tissue before
fixation
Honing ▪ removal of gross nicks on the knife edge (coarse hoaning)
Clearing becomes ▪ water not removed Repeat dehydration with
▪ removal of blemishes and irregularities from knife edge milky as soon as tissue completely (incomplete absolute alcohol, then clear
▪ Direction: heel to toe is placed in it dehydration) again
Stropping ▪ for sharpening ▪ incomplete removal of ▪Trim the blocks down nearest
On trimming, tissue clearing agent due to to the tissue
▪ removal of burr or irregularities during honing
smells of clearing insufficient impregnation ▪Melt the remaining wax on
▪ final polishing of the knife edge agent embedding oven
▪ Direction: toe to heel (reverse of honing) ▪Repeat paraffin impregnation
Tissue is opaque ▪ insufficient clearing Repeat clearing
STROPPING section cutting is
Hone: a natural stone or hard grinding surface for sharpening a knife difficult due to
presence of alcohol
Tissue shrinks away ▪ insufficient dehydration Repeat whole procedure
from the wax when
 trimmed
Tissue is soft when ▪ incomplete fixation
block is trimmed
▪ incomplete impregnation
Airholes found on
▪ contaminated wax
tissue during trimming
▪ block not cooled rapid
(appear crystalline)
enough
Moist & crumbled ▪ insufficient paraffin
Paraffin block after impregnation
cooling
▪ surfaces and edges of block
are not parallel
▪ horizontal surface of the
block is not parallel to the
knife
Sections fail to form
▪ paraffin wax is too hard
ribbons
▪ knife is tilted too much
▪ sections are too thick
▪ knife is dull
Sections roll up on ▪ knife is dull
cutting so that they ▪ knife is tilted too much
adhere and get broken
▪ knife edge is dirty
and against the knife
edge
▪ blunt or dull spot on knife
(irregular knife edge)
Ribbon is curved,
crooked or uneven ▪ edges of the block are not
instead of straight parallel but round or wedge-
shaped
▪ knife is not parallel to the
block
▪ paraffin is impure
▪knife is blunt or dull
▪ paraffin block is warm and
soft
Sections are ▪ knife edge is coated with
compressed, wrinkled paraffin
or jammed ▪ sections are too thin
▪ microtome set screw is
loose
▪ tilt of knife is too vertical
▪incomplete dehydration,
clearing and infiltration of
tissue with wax
Sections are torn and ▪ paraffin is warm and soft
crumble when cut ▪ knife is blunt
Sections are squashed ▪ Bevel of knife is lost due to
width of each section incorrect sharpening
less than that of the
block
▪ bubble or dirt formed in the
embedding medium
A Hole is formed in ▪ hard spot in tissue possibly
the section due to calcium
▪ tilt of knife is too great or
bevel is not cleared, hence
object is compressed against
the knife edge
Sections of unequal
▪ clamp set screw on the knife
thickness are
or blockholder is loose
produced
▪ blocks are too large
▪ blocks are too hard
▪ static electricity due to low
Sections adhere to the atmospheric humidity
knife or other parts of ▪ knife edge is dirty
the machine ▪ knife edge is dull
▪ knife tilt is too great
▪ nicks/damage on the knife
Ribbon is split or edge
lengthwise vertical
▪ dirty embedding medium
scratches are seen on
section ▪ knife edge is dirty
▪ tilt of the knife is too great
▪ knife tilt is too great
Sections are lifted
▪ knife is dull
from the knife on
upstrokes ▪ paraffin is too soft r RT is
warm
▪ tilt of knife is insufficient, ▪ increase the tilt
Repeat fixation Resistance is felt on paraffin block is therefore
the lower part of compressed against the base
Reembed in freshly filtered section during cutting of knife towards the end of
wax the stroke
Horizontal or parallel ▪knife edge vibrates due to: ▪ treat with phenol during
lines or forrows across
a) hardness of the tissue processing or collodionize
the sections/chatters b) tilt of the knife is too great
Repeat paraffin impregnation,
are seen, forming thin
reembed
and thick zones
▪Retrim the block ▪ knife is blunt ▪ tighten adjusting and locking
▪ knife isn’t clamped properly screws
▪ tilt of knife is too great ▪ increase tilt
▪Re adjust and reorient the Section cut is
block sometimes thin, ▪ knife or blockholder is loose
sometimes thick ▪ knife tilt is too small that
▪Coat horizontal edge of block block is compressed by bevel
with wax of lower MP and section isn’t cut
▪Reduce the tilt of knife Knife makes a hard ▪ tilt of knife is too slant or ▪ readjust the angulation of
▪Readjust the thickness metallic scraping or too big the knife
ringing sound on ▪ tissue is too hard ▪ take fresh block
backstroke, when
▪ knife blade is too thin ▪ change the knife
sectioning
Sharpen the knife
Frozen tissue crumbles ▪ freezing isn’t adequate Refreeze the tissue
Reduce the tilt
and comes off the
Clean the knife edge
blockholder when cut
Frozen tissue chips ▪ tissue is frozen too hard ▪ warm the tissue with fingers
into fragments when
▪Adjust the knife so that knife
cut
edge will present formly sharp
edge to the block or sharpen
▪Retrim the block DIFFICULTIES DERIVED FROM THE TISSUES
▪Readjust the knife and the blood clot, very hard in routine block before cutting, effect with a firm sharp
block cervix, thyroid processing stroke. Use chloroform as clearing agent. Use
tetrahydrofuran for dehydrating agent.
▪Repeat impregnation using tissue
pure wax fatty tissues soft blocks and shredded Cut thin 2mm, and impregnate with wax in
(subcutaneous tissue vacuum bath
tissue, breast,
lipoma)
▪Resharpen the knife
brain and very hard and brittle (if Use chloroform and vacuum impregnation
▪Cool the block on ice water using xylol for with lymph nodes
lymph nodes
until firm dealcoholization)
▪Clean the knife edge soft tissue Tend to expand more set and cool the wax block 10-12%shrinkage..
when floated out Split the outer rim of wax with a dissecting
▪Readjust the thickness of needle.
section
▪Thighten the screw CELLOIDIN SECTIONS 徐智慧/
▪Reduce the tilt
▪ 10-15u in thickness
▪remove paraffin with clearing
agent, pass thru decreasing ▪ don’t require hardening by chilling before cutting
grade of alcohol, then repeat ▪ use sliding microtome
dehydration, clearing and ▪ use wet method to avoid dehydration and shrinkage
embedding
▪ cool and harden paraffin in
FROZEN SECTIONS
ice water for ¼ to ½ hour.
2 METHODS OF PREPARATION
▪ sharpen the knife
Cold ▪ knife ▪ CO2 (Freezing ▪ filter paper soaked in gum
▪ resharpen, using a knife back
knife -40 to -60C agent) syrup on microtome stage
or automatic knife sharpener
▪ tissue ▪ different temp ▪ apply short burst of CO2
-5 to -10C between knife ▪ 3-5mm thick
▪ reembed in freshly filtered ▪ environment and tissue (latter ▪ 5 sec interval
0 to -10C is colder) ▪ dew line: the point at which
wax if necessary
▪ once embedded in paraffin sections may be cut at 10 u.
wax, decalcification is Cryostat Near -20 C Advantages: Disadvantage:
impractical use a base sledge ▪ (-5 to -15C) ▪ staining (fat ▪ sections don’t form ribbons
microtome with a wedge knife brain, lymph nodes, demo by oil red but stick to the knife blade
▪ reduce the tilt liver, spleen, kidney, O, silver (remove with camel’s hair
testis, uterine, impregnation, brush)
tumor, thyroid CNS methods) ▪ lack of embedding mass,
▪ (-15 to -20C) ▪ indispensable distorted structural details
▪ tighten screw Muscle, conn tissue, for rapid during cutting
pancreas, uterus, diagnosis during ▪ staining of unfixed tissue is
cervix, skin without operation
▪ cut blocks into smaller rarely unsatisfactory
fragments fat, nonfatty breast ▪ enzyme demo ▪ produce freezing artifact
tissue, ovary,
▪ soften blocks in detergent or
prostate, tongue, gut
phenol
▪ (-35C)
Breathe out or blow gently on
Fatty tissue, fatty
the block and knife to break up
breast, omental
static electricity or boil in water
in room to increase the
humidity
▪ sharpen the knife
▪ reembed in filetered wax
▪ clean the knife edge with
xylol
▪ cool paraffin wax in ice water
 STAINING 徐智慧/ COMPOSITION OF DYES
 Formation of colors of different tissues and cells Alum Hematoxylin a) Ehrlich’s hematoxylin
-LEEUWENHOEK (saffron) ▪ For progressive ▪ Regressive staining
-GOPPERT, COHN (carmine) staining ▪ for muco
▪ Counterstained with polysaccharide, cartilage,
-GERLACH (sel. Nuclear stain) Hematoxylin
congo red & safranin. cement lines of bones
▪ Low affinity for b) Harris Hematoxylin
PURPOSES OF STAINING ▪ Salt lakes color: Blue
tissue, must use ▪ for routine nuclear staining
▪ Blueing: process of
▪ render different tissue constituents (more visible) mordants (alum, ▪ for exfoliative cytology
passing the tissue to
▪ easier optical differentiation for ID of cells and tissue iron, chromium,
alkaline solution to ▪ for staining sex
copper salts)
▪ display various affinities neutralize acid. chromosomes
▪ Extraction from
▪ display physical char and structural relationships heartwood of ▪ Cold water (slows c) Cole’s hematoxylin
down) ▪ for routine purposes
Mexican Tree:
Hematoxylin ▪ warm water ▪ used in sequence with
METHODS TO OBTAIN STAINING REACTION:
(accelerates) Celestine blue
▪ Capillary osmosis campechianum
▪ Hematin ▪ Very cold water d) Mayer’s hematoxylin
▪ Solubility Active coloring (<10C, produce pink ▪ used in Celestine blue
▪ Adsorption agent, oxidized artifacts) ▪ for nuclear staining
▪ Absorption hematoxylin a) Weigert’s hematoxylin
▪ Ripening ▪ std. for lab
TYPES OR METHODS OF STAINING Oxidation process of ▪ muscle fiber demo
hematoxylin to ▪ conn.tissue demo
Direct Use simple aqueous or alcoholic solution of the dye hematin.
Indirect ▪Mordant (link/bridge to form color) (exposure of stain to
b) Heidenhain’s hematoxylin
▪ cytological stain
▪Accentuator (accelerate/hasten the speed of staining) air and sunlight)
Iron Hematoxylin
OXA: ▪ for regressive staining of
Progressive Not washed or decolorized ▪ Mordants: ferric thin sections
-H2O2
▪ less favorable than regressive. Difficultly of producing -HgCl2 ammonium sulfide ▪ for nuclear and cytoplasmic
intense cell structure, diffused and obscured effect (iron alum) inclusion (chromatin,
-KMnO4

From plants, animal

(for wool & cotton)
Regressive Gram staining -Na perborate chromosome, nucleoli,
-NaI centrosome, mitochondria)

NATURAL
excess stain is decolorized
c) Phospho tungstic acid
▪differentiation/decolorization: selective removal of excess hematoxylin (PTAH)
stain from tissue ▪ for structures in paraffin,
Counterstaining Apply different color/stain to provide contrast and celloidin, frozen section
background. Cochineal Dyes
CYTOPLASMIC ▪ old histologic dye
▪RED ▪Yellow ▪Green ▪ from extraction of
-Eosin Y,B -Picric acid -Light green SF female cochineal bug
-Phloxine B -Orange G -Lissamine green (coccus cacti)
-Rose Bengal ▪ treated with alum
NUCLEAR to produce carmine
dye
▪RED ▪Blue
▪ combination with
-Neutral red -Methylene blue picric acid/
-Safranin red -Toluidine blue picrocarmine is used
-carmine -celestine blue for
-hematoxylin neutropathological
Metachromatic Use specific dye (metachromasia) study
Thiazine and triphenylmethane group ▪ combination with
-methyl violet/crystal violet AlCl3/ best’s carmine
is used for glycogen
-cresyl blue
demo
-safranin
Orcein Dye
-bismarck brown
▪ vege dyes from
-basic fuchsin
lichens
-methylene blue ▪ colorless
-thionine ▪ combination with
-toluidine blue ammonia and
-Azure A,B,C exposure to air
Microanatomical Demostrante general relationship of tissue and cell without produce blue and
emphasizing the inclusion bodies violet
▪Cytoplasmic staining
-doesn’t differentiate tissue structure in general ▪ Chromophores Dyes:
(from C6H6 hydrocarbon benzene)
Synthetic dyes/ coal tar dyes /Aniline dyes

-produce visible a) Acid Dyes

▪Negative staining
colors ▪ acid: coloring substance
-demonstrate bacterial morphology in black background
(india ink) ▪ Chromogens ▪ base: sodium
Metallic Demonstrate tissue elements not by stains but by colorless -benzene b) Basic Dyes
Impregnation solutions of metallic salts. compounds with ▪ acid: sulfuric, acetic or HCl
*adsorption chromophore ▪ basic: coloring substance
The most valuable metal: gold (gold chloride) and silver ▪ Auxochrome c) Neutral dyes
(silver nitrate) -auxiliary radical ▪ Acid aq + basic aq
▪ Explosive (props of ▪ for nucleus and cytoplasm
electrolytic
▪ avoid silver glasswares ▪ soluble in alcohol
dissociation)
▪ don’t expose to sunlight ▪ insoluble in water
-alter the shades
▪avoid metallic instruments of dye
Vital ▪ Selective staining of living cell constituents -give the props of
▪demonstrate cytoplasmic structures by phagocytosis of forming salts
SUPRAVITAL: dye particle
-neutral red ▪nucleus is resistant to vital stain
-janus green
INTRAVITAL STAINING
-tryphan blue
inject the dye into any part of animal body
-nile blue
(intravenous/intraperitoneal/subcutaneous)
-thionine
SUPRAVITAL STAINING
-toluidine blue
stain living cells immediately
DIFFERENT STAINS USED IN HISTOPATHOLOGY Fungi Ziehl neelsen
Aniline blue Cytoplasmic, epithelial section Nocardia Gram staining
counterstain Amoeba Heidenhain’s iron hematoxylin
Basic Plasma stain acid fast organism, mitochondria, Inclusion bodies/ negri Schleifsten’s method
fuchsin smooth muscle Calcium Calcium Prussian blue
Fuelgen’s + schiff’s reagent: detect Urates De galantha stain
aldehydes
Van gieson’s : conn.tissue, mucin, ROUTINE HEMATOXYLIN STAINING PROCEDURE
elastic tissue Xylol I Removes
Bismarch Contrast stain gram’s technique, acid fast & 5 min
Xylol II paraffin
brown papanicolau method, diphtheria DEPARAFFINIZATION
Acetone-alcohol
organism 3 min Removes xylol
Acetone
Carmine Chromatin stain smear prep…
Alcohol 95%
Best carmine solution for glycogen
Alcohol 80%
Mucicarmine for mucin HYDRATION 3 min Hydrates
Alcohol 70%
Celestine Resistant to strong acid for routine staining of fixed sections,
Alcohol 50%
blue dyes +alumH = good nuclear staining
Wash with distilled water
Crystal Nuclear/chromatin stain stain amyloid in frozen section, stain
STAININIG Hematoxylin 5 min Stains nuclei
violet platelet in blood
Rinse with water
Eosin Eosin B : bluish deeper Stain conn tissue and cytoplasm
red color Routine: Hematoxylin – Eosin – DECOLORIZATION Acid alcohol 1 dip Decolorize
Eosin Y : yellowish methylene blue Ammonia water
BLUEING Alcohol 80% Dip to blue Blues nuclei
Giemsa Stain blood to differentiate Alcohol 95%
stain leukocyte COUNTERSTAINING Eosin 8 min Stains cytoplasm
Malachite Weakly basic, contrast Ascaris eggs, erythrocyte, Alcohol 95%
Dehydrates and
green stain, decolorizer, Alcohol 95%
DEHYDRATION 3 min removes excess
counterstain Acetone
stain
Methyl Chromatin stain, Gives Chromatin green (w/acid) Acetone-xylol
green false positive with mucin Xylol I Clears out
CLEARING 5 min
secretion Xylol II alcohol
Methylyne Basic nuclear stain, Fresh sputum, malignant cells,
blue plasma cells, cytological bacterial stain, milk grading, RESULTS
evaluation diphtheria, nervous tissue, Basophil cytoplasm, plasma cells, osteoblast Purplish
Methylene Metachromatic dye Leukocytes’s nuclei: reddish purple Calcium and calcified bone Purplish blue
violet with methylene blue Cartilage Pink/light blue to dark blue
Orcein Dermatological study Excellent for elastic fibers Cement line of bones Blue (ehrlich)
Picric acid Contrast stain Acid fuchsin, Collagen, osteod Light pink
Cytoplasmic stain conn.tissue (van gieson) Cytoplasm Pink
Counterstain crystal violet Decalcified bone matrix Deep pink
Tissue fixative Karyosome Dark blue
Decalcifying agent Muscle fibers, thyroid colloid, thick elastic fibers Deep pink
Prussian Colored salt of ferric Manufacture of paints Nuclei Blue to blue black
blue ferrocyanide RBC, Eosinophil granules, keratin Bright orange red
Microanatomical color Circulatory system
contrast DIFFICULTIES IN STAINING
Toluidine Nuclear stain Fixed tissue Stains on the skin ▪ poor technique
blue Substitute Thionine in frozen section
Recommended for: -nissl granules ▪ health hazards
-chromophilic bodies Flake or float off tissue ▪ dirty or greasy slide
section ▪ inadequate fixation on the slide
COMMONLY USED SPECIFIC TISSUE STAIN ▪ torn section
Routine histologic study H&E ▪ sections contain air bubbles
Conn. Tissue/ collagen Methylene blue ▪ inadequate infiltration
reticulum Gold method ▪ albumin adhesive is too old
Elastic tissue Orcein
▪ unthorough spreading
Muscle tissue Van gieson’s
Section doesn’t take up the ▪ insufficient/improperly ripened hematoxylin
fibrin H&E
Amyloid Methyl violet
stain ▪ impurities in the dye/water solvent
Glycogen Best carmine ▪ inadequate paraffin washing off
Mucin Meyer’s mucicarmine ▪ loss of staining ability (ppt)
Acid mucopolysaccharide Toluidine blue Sections don’t appear clear ▪ xylol should be replaced
Nissl bodies H&E under microscope ▪ water in absolute alcohol
Neurons, axons, neurofibrils Bielschowsky’s technique ▪ moisture in the coverslip
Myeline sheath Sudan black ▪ too much egg albumin on the slide
Astrocytes Mallory’s PTAH ▪ decolorizer wasn’t completely removed
Argentaffin granules Masson Fontana silver method
Phospholipids Baker’s technique RESTAINING OLD SECTIONS (OLD/BLEACHED/FADED)
Neutral fat Sudan black Xylol (heat until mounting media bubbles) 24 hr
Fatty acids Lillie’s sulfuric acid nile blue Remove coverslip by dissecting needle
Hb Amido black Xylol to remove remaing mounting media 30 min
Hemosiderin Prussian blue Wash with water
Hematoidin Gmelin’s Potassium permanganate 5-10 min
Bile pigment Gmelin’s Wash with water
Hemofuchsin pigment Mallory’s fuchsin stain 5% oxalic acid until decolorized 5 min
Melanin Masson Fontana Wash with water 5 min
Bacteria Gram staining Restain
Actinomyces Brown breen’s Mount
Acid fast Ziehl neelsen
Spirochetes Giemsa stain
MOUNTING OF SECTIONS 徐智慧/ SPECIAL PROCESSING TECHNIQUE
Purpose:  when chemical constituents of tissues shouldn’t have been altered/removed/
▪ protect spn from physical injury displaced
▪ protect section from bleaching or deterioration Frozen Section  most ideal tissue prsv. To avoid complete or partial loss of
enzyme
▪ preserve slide for permanent keeping
General Principle
MOUNTING MEDIUM  syrupy fluid applied between section and coverslip,
▪ Quenching/freezing = produce instant cessation of cellular activities, prevent
preventing movement of the coverslip
chemical alteriation
CHARACTERISTICS OF GOOD MOUNTING MEDIUM ▪ rapid freezing = prevent ice crystal artifacts formation
▪ Ref.Index : near to the glass 1.518 ▪ freezing agents: Liquid nitrogen
▪ shouldn’t dry quickly ▪ isopentane, pentane, propane = cooled to very low temp, to retain fluidity
▪ shouldn’t dissolve out or fade tissue section
▪ should not cause shrinkage and distortion METHODS TO AVOID CHEMICA FIXATION OF BLOCKS
▪ should set hard FREEZE DRYING ▪ 2mm tissue + freezing agent + liquid nitrogen
▪ shouldn’t have granularity/ cracking -quenching/rapid ▪ solidification 2-3sec = prevent ice crystal artifacts
freezing ▪ high vacuum drying apparatus
▪ should be miscible with xylene or toluene
-dessication
▪ should be nonreactive, no pH or color change ▪ water sublimation
(removal of water)
▪ dessication 24-48hr
CLASSIFICATION ▪ infiltration, impregnation in vacuum embedding oven
Temporary/ Aqueous ▪ for water miscible prep ▪ sectioning
▪ made of gelatin to solidify medium ▪ staining
▪ made of glycerol to prevent cracking
▪ made of sugar to increase ref. index DISADVANTAGE ADVANTAGES:
▪ made of a prsv. Solution -time consuming -min. shrinkage
-expensive -fresh tissue
Permanent/ Resinous ▪ for dehydrated prep and cleared prep
-diff.to section -min. chemical change
▪ natural/synthetic -brittle -less displacement
-not for routine -for enzyme studies
PERMANENT MOUNTING MEDIA FREEZE ▪ fixation with rossman’s fluid or osmium tetroxide in 1%
Canada balsam ▪ from Canadian tree (abus balsamea) SUBSTITUTION acetone 4-6 days (-60 to -70C)
▪ dissolved in xylene -rapid freezing ▪ infiltration, embedding
▪ recommended for whole mounts and thick section -subsequent ▪ more economical
▪ darken slightly with age infiltration,
▪ recommended for routine
DPX/ Kirkpatrick and ▪Ref. index = 1.52 embedding
lendrum ▪ recommended for small tissue section
FRESH FROZEN ▪ maintain fresh and solid state
TISSUE
Clarite X ▪ most widely used in north America SECTIONING
▪Synthetic resin
▪ ref index = 1.544
XAM ▪ synthetic resin mixture in xylene
▪ pale yellow/colorless
▪ dries quickly without retraction, prsv. Stains well

SEMIPERMANENT OR TEMPORARY MOUNTING MEDIA

Water ▪ low ref index
▪ good visibility
▪ least permanent
▪ not for OIO
Glycerin ▪ last for months
▪ ref index 1.46
Glycerin jelly ▪ good for fat stains (ref index 1.4-1.47)
Farrant medium ▪ ref index 1.44
Von apathy’s gum syrup ▪ for methylene blue stained nerve prep
▪ gen.purpose aqueous mountant
▪ ref index 1.52
Brun’s fluid ▪ recommended for mounting frozen sections
from water
Levulose/ fructose ▪ high ref index 1.5
▪ doesn’t leach metachromatic stain
Mineral oil/ liquid paraffin ▪ for romanowsky stained smear

SEALING
RINGING  process of sealing the margins of coverslip to prevent escape of fluid.
Ringing media:
-paraffin wax -kronig’s/Dunoyer’s mixture
-nail polish -Durofix (cellulose adhesive)

REASONS FOR RESTAINING SECTION

-faded section
-superimposed staining

BROKEN SLIDES
-unfragmented slide, unimportant section  may be reassembled
-vital part section, unavailable replacement  transfer to another slide
 DECALCIFICATION 徐智慧/
 Process of removing calcium salts or lime salts and its deposits from hard WAYS TO DETERMINE THE EXTENET OF DECALCIFICATION
tissue for softening of tissue to facilitate embedding and sectioning. Physical/mechanical test ▪Touching/bending tissue with fingers
FIXATION  DECALCIFICATION  IMPREGNATION ▪determine consistency
X-RAY or radiological method ▪can detect smallest focus of Ca
**Inadequate decalcification = poor cutting of hard tissue, damage to knife edge ▪ very expensive
Chemical method ▪ simple, reliable, convenient
Tissues for decalcification:
-bones CLOUDY:
-teeth -Incomplete
-atheromatous aorta -presence of Ca
-calcified tuberculous foi NOT CLOUDY:
-complete
BEFORE CALCIFICATION -no more Ca
▪ cut tissue into small pieces 5 mm = permit complete penetration ,
▪ adequate fixation
▪ place tissue in buffered neutral formalin for 2-4 days or helly’s fluid 15-24hr

GOOD DECALCIFYING AGENT:

▪ can remove calcium salts completely
▪ doesn’t produce destruction of cells

DECALCIFYING AGENTS
Acid ▪ most widely The rate is affected Nitric acid
used by: HCl
▪ stable ▪ tissue structure Formic acid
▪ easily available ▪ temp Trichloroacetic
▪ volume and conc. acid
▪ relatively
Sulfurous acid
inexpensive Of solution
Chromic acid
▪ external factor
Citric acid
(accelerate)
Chelating Combines Ca2+ to Advantage: ▪ EDTA/
agents form soluble non -forms min. Versene
ionized complex, histological artifacts
facilitate removal Disadvantage:
of calcium. -slow reacting
Ion exchange Ammonium form Advantages:
resin of plystyrene resin Cellular detail is well
▪ remove calcium preserved
ions from formic
acid , increase
solubility
Electrical ▪ Ca2+ are Advantage:
ioinization/ attracted to ▪Faster due to heat
electrophoresis negative electrode, and electrolyte
then removed reaction
from decalcifying ▪ recommended for
solution. small bone
fragments
Disadvantage:
Not well preserved

NITRIC ACID: most common, very rapid, min. distortion, inhibits nuclear staining,
destroy tissue
Aquous nitric acid
12-24 hr ▪ rapid ▪ prolonged may
solution 10%
Formol nitric acid 1-3 days ▪ for urgent biopsy distort tissue
Perenyi’s fluid 2-7 days ▪ no maceration ▪ slow
Phloroglucin nitric ▪ poor nuclear
12-24 hr ▪ most rapid
acid staining
HCl : slow, great distortion, good nuclear staining
FORMIC ACID: Moderate, beter nuclear staining, for postmortem studies. ▪
fixative + decalcifying agent in one ▪ slow
Formic acid 3-14 ▪ excellent nuclear ▪ slow
sodium citrate days staining ▪ not routine
TRICHLOROACETIC ACID : 4-8 days, good nuclear staining, weak, not for dense
tissue
SULFUROUS ACID: very weak, only for min. bones
CHROMIC ACID/FLEMMING’S FLUID: fixative + decalcifying agent, inhibit nuclear
staining
CITRIC ACID-CITRATE BUFFER 4.5pH: use chloroform as prsv., excellent nuclear
and cytoplasmic staining
VON EBNER’S FLUID: good cytologic staining, for teeth and small bones

DEGREE OF DECALCIFICATION:
▪ shortened/prolonged = unsatisfactory
▪ prolonged = maceration, destruction
▪ underdecalcification = interfere normal cutting