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HISTOPATHOLOGY SUMMARY
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SECTIONING Sectioning : cutting tissue uniformly into a thin slice/section with the aid of a machine to facilitate the studies under the microscope. Microtome : a machine/instrument designed for actual cutting of thin section.
KINDS OF MICROTOME
Sliding (celloidin-embedded section)
Base-sledge type - 2 movable pillars holding the knife - chuck/block holder is set on a heavy metal base - block holder is raised towards the knife
Std. sliding type - The block remains stationary - the knife is moved backward and forward during sectioning
Rotary - For paraffin-embedded sections - most common type used for routine & research laboratories
Freezing - For unembedded tissue - a simple lever operated valve release a rapid, intermittent burst of CO2 to freeze the block holder
▪ For rapid Dx
▪ For histological demo of fat
▪ For the study of neurological structure
▪ For the study of sensitive tissue constituent which are
damaged/ destroyed by heat
Rocking - For serial sections of large blocks of paraffin-embedded tissue - produce 60-90sections with ease
▪ Lower arm : resting on pivots and supporting column
▪ Upper arm : carry the block holder on one end by a screw
Ultra thin sectioning microtome
- For electron microscopy
MAJOR PARTS OF A MICROTOME
Block Holder/ chuck Holds the tissue block in place
Knife carrier and knife Actual cutting of tissues
Pawl, ratchet feed wheel, adjs.screw
Line up the tissue in proper position to the knife, Adjust proper thickness of the tissue for successive sections
CARE OF THE ROTARY MICROTOME
▪ after cutting, brush away with soft brush: all accumulated paraffin and tissue
▪ wipe clean all metal parts with xylol
▪ avoid continuous application of xylol to the rest of the machine (can remove
the painted finish)
▪ Dry the machine carefully especially the knife holder
▪ keep the machine well oiled to prevent rust formation
▪ keep the moving parts of microtome oiled
▪ cover the microtome when not in use (prevent accumulation of dust and dirt)
(may interfere the sectioning) MICROTOME KNIVES
PARTS OF MICROTOME KNIFE
Cutting edge Cutting facet, made of good quality steel. Able to cut good section from a paraffin wax block without any serration note on examination.
Wedge
Knife back Spring-loaded semicircular sheet of metal slipped on the knife
Bevel angle Angle between the cutting edge: 27-32
Wedge angle Angle formed by the sides of the wedge knife (5-14)
Clearance angle Angle between cutting facet (5-15)
KINDS OF MICROTOME KNIVES
Plane concave 250mm, 1 side is flat, 1 side is concave
Biconcave 120mm, both sides concave
Plane wedge 100mm, both sides straight
CARE OF MICROTOME KNIFE
Honing ▪ removal of gross nicks on the knife edge (coarse hoaning)
▪ removal of blemishes and irregularities from knife edge
▪ Direction: heel to toe
Stropping ▪ for sharpening
▪ removal of burr or irregularities during honing
▪ final polishing of the knife edge
▪ Direction: toe to heel (reverse of honing)
STROPPING Hone: a natural stone or hard grinding surface for sharpening a knife
TYPES OF HONES
Belgium yellow/ hone ▪ for manual sharpening
▪ usually gives best result
Arkansas ▪ gives more polishing effect
Fine carborundum ▪ much coarser
▪ used for badly nicked knives
Plate glass hone ▪ use diamanthine for final polishing
Machine hone ▪ glass disc or wheel driven by electric motor
COMMON LUBRICANT USED FOR HONING - mineral oil - clove oil - xylene - liquid paraffin - soapy water CARE AND USE OF MICRTOME KNIVES
▪ perfect edge: junction of the smooth plane surface 14°
▪ use fine yellow Belgian water stone to remove large nicks
▪ size 8’’ x 3’’
▪ keep the knife flat when being honed
▪ finish the edge on a leather or linen strop
▪ wipe the knife clean with soft cloth before and after stropping strokes
▪ use gentle pressure when stropping
▪ avoid speed in stropping
▪ leather strop require oiling before use. Use castor oil or vege oil to treat the
strop applied at the back of the strop (not on the surface)
▪ mineral oil destroys the leather strop, done allow to come in contact with the
strop
▪ wax shouldn’t come in contact with the strop
TYPES OF TISSUE SECTIONING
PARAFFIN SECTIONS
▪ Prep: trim the block until all sides are parallel
▪ Adhesive: for entailing significant exposure of section to acids and alkalis, but
cannot be used for protein histochemical investigation
Mayer’s albumin Egg white + glycerin + thymol crystal
Dried albumin Dried albumin + NaCl + thymol crystal
Gelatin Gelatin + water + glycerol +phenol crystal
Gelatin chrome alum
Can be added to water bath.
Starch paste Powdered starch + HCl + thymol crystal
Plasma Outdated blood stored in blood banks
▪ Actual Sectioning -nervous tissue, lymph nodes : slow, gentle motion -the harder the tissue, the harder & cooler the block should be
▪ Floating out bath
45-50C or 6-10C lower than the melting point of paraffin
▪ Drying of sections
-wax oven 56-60C (2hours) -incubator 37C (overnight) -hot plate 45-55C (30-45 min) -blower type electric slide dryer 50-55C (20-30min) -bunsen flame until the wax melts -nervous tissue: incubation overnight 37C
DIFFICULTIES ENCOUTERED IN SECTION CUTTING
FAULTS REASON REMEDY
Brittle/hard tissue
▪ prolonged fixation
▪ prolonged dehydration
▪ prolonged clearing
▪ prolonged paraffin infiltration in over heated paraffin oven
▪ drying out of tissue before fixation
Soften tissue by soaking oil in a smaller dish or bowl (w/water) with detergent, phenol or molliflex
Clearing becomes milky as soon as tissue
is placed in it
▪ water not removed
completely (incomplete dehydration)
Repeat dehydration with absolute alcohol, then clear again
On trimming, tissue smells of clearing
agent
▪ incomplete removal of
clearing agent due to insufficient impregnation
▪Trim the blocks down nearest
to the tissue
▪Melt the remaining wax on embedding oven
▪Repeat paraffin impregnation
Tissue is opaque section cutting is difficult due to
presence of alcohol
▪ insufficient clearing Repeat clearing
Tissue shrinks away from the wax when
▪ insufficient dehydration Repeat whole procedure
trimmed
Tissue is soft when block is trimmed
▪ incomplete fixation Repeat fixation
Airholes found on tissue during trimming
(appear crystalline)
▪ incomplete impregnation
▪ contaminated wax
▪ block not cooled rapid enough
Reembed in freshly filtered wax
Moist & crumbled Paraffin block after
cooling
▪ insufficient paraffin
impregnation
Repeat paraffin impregnation, reembed
Sections fail to form ribbons
▪ surfaces and edges of block are not parallel
▪ horizontal surface of the block is not parallel to the knife
▪ paraffin wax is too hard
▪ knife is tilted too much
▪ sections are too thick
▪ knife is dull
▪Retrim the block
▪Re adjust and reorient the block
▪Coat horizontal edge of block
with wax of lower MP
▪Reduce the tilt of knife
▪Readjust the thickness
Sections roll up on cutting so that they
adhere and get broken and against the knife
edge
▪ knife is dull
▪ knife is tilted too much
▪ knife edge is dirty
Sharpen the knife Reduce the tilt Clean the knife edge
Ribbon is curved, crooked or uneven instead of straight
▪ blunt or dull spot on knife
(irregular knife edge)
▪ edges of the block are not
parallel but round or wedge-shaped
▪ knife is not parallel to the block
▪ paraffin is impure
▪Adjust the knife so that knife
edge will present formly sharp edge to the block or sharpen
▪Retrim the block
▪Readjust the knife and the
block
▪Repeat impregnation using pure wax
Sections are compressed, wrinkled
or jammed
▪knife is blunt or dull
▪ paraffin block is warm and
soft
▪ knife edge is coated with paraffin
▪ sections are too thin
▪ microtome set screw is loose
▪ tilt of knife is too vertical
▪Resharpen the knife
▪Cool the block on ice water
until firm
▪Clean the knife edge
▪Readjust the thickness of
section
▪Thighten the screw
▪Reduce the tilt
Sections are torn and crumble when cut
▪incomplete dehydration, clearing and infiltration of tissue with wax
▪ paraffin is warm and soft
▪ knife is blunt
▪remove paraffin with clearing agent, pass thru decreasing grade of alcohol, then repeat dehydration, clearing and embedding
▪ cool and harden paraffin in
ice water for ¼ to ½ hour.
▪ sharpen the knife
Sections are squashed width of each section less than that of the
block
▪ Bevel of knife is lost due to incorrect sharpening
▪ resharpen, using a knife back or automatic knife sharpener
A Hole is formed in the section
▪ bubble or dirt formed in the embedding medium
▪ hard spot in tissue possibly
due to calcium
▪ reembed in freshly filtered wax if necessary
▪ once embedded in paraffin
wax, decalcification is impractical use a base sledge microtome with a wedge knife
Sections of unequal thickness are
produced
▪ tilt of knife is too great or
bevel is not cleared, hence object is compressed against the knife edge
▪ clamp set screw on the knife
or blockholder is loose
▪ blocks are too large
▪ blocks are too hard
▪ reduce the tilt
▪ tighten screw
▪ cut blocks into smaller fragments
▪ soften blocks in detergent or
phenol
Sections adhere to the knife or other parts of
the machine
▪ static electricity due to low atmospheric humidity
▪ knife edge is dirty
▪ knife edge is dull
▪ knife tilt is too great
Breathe out or blow gently on the block and knife to break up static electricity or boil in water in room to increase the humidity
Ribbon is split or lengthwise vertical
scratches are seen on section
▪ nicks/damage on the knife
edge
▪ dirty embedding medium
▪ knife edge is dirty
▪ tilt of the knife is too great
▪ sharpen the knife
▪ reembed in filetered wax
▪ clean the knife edge with
xylol
Sections are lifted from the knife on
upstrokes
▪ knife tilt is too great
▪ knife is dull
▪ paraffin is too soft r RT is warm
▪ cool paraffin wax in ice water
Resistance is felt on the lower part of
section during cutting
▪ tilt of knife is insufficient, paraffin block is therefore compressed against the base of knife towards the end of the stroke
▪ increase the tilt
Horizontal or parallel lines or forrows across the sections/chatters are seen, forming thin
and thick zones
▪knife edge vibrates due to:
a) hardness of the tissue b) tilt of the knife is too great
▪ treat with phenol during
processing or collodionize
Section cut is sometimes thin, sometimes thick
▪ knife is blunt
▪ knife isn’t clamped properly
▪ tilt of knife is too great
▪ knife or blockholder is loose
▪ knife tilt is too small that block is compressed by bevel
and section isn’t cut
▪ tighten adjusting and locking screws
▪ increase tilt
Knife makes a hard metallic scraping or
ringing sound on backstroke, when
sectioning
▪ tilt of knife is too slant or too big
▪ tissue is too hard
▪ knife blade is too thin
▪ readjust the angulation of the knife
▪ take fresh block
▪ change the knife
Frozen tissue crumbles and comes off the
blockholder when cut
▪ freezing isn’t adequate Refreeze the tissue
Frozen tissue chips into fragments when
cut
▪ tissue is frozen too hard ▪ warm the tissue with fingers
DIFFICULTIES DERIVED FROM THE TISSUES
blood clot, cervix, thyroid tissue
very hard in routine processing
block before cutting, effect with a firm sharp stroke. Use chloroform as clearing agent. Use tetrahydrofuran for dehydrating agent.
fatty tissues (subcutaneous tissue, breast, lipoma)
soft blocks and shredded tissue
Cut thin 2mm, and impregnate with wax in vacuum bath
brain and lymph nodes
very hard and brittle (if using xylol for dealcoholization)
Use chloroform and vacuum impregnation with lymph nodes
soft tissue Tend to expand more when floated out
set and cool the wax block 10-12%shrinkage.. Split the outer rim of wax with a dissecting needle.
CELLOIDIN SECTIONS
▪ 10-15u in thickness
▪ don’t require hardening by chilling before cutting
▪ use sliding microtome
▪ use wet method to avoid dehydration and shrinkage FROZEN SECTIONS
2 METHODS OF PREPARATION
Cold knife
▪ knife -40 to -60C
▪ tissue
-5 to -10C
▪ environment 0 to -10C
▪ CO2 (Freezing agent)
▪ different temp
between knife and tissue (latter is colder)
▪ filter paper soaked in gum syrup on microtome stage
▪ apply short burst of CO2
▪ 3-5mm thick
▪ 5 sec interval
▪ dew line: the point at which sections may be cut at 10 u.
Cryostat Near -20 C
▪ (-5 to -15C) brain, lymph nodes, liver, spleen, kidney, testis, uterine, tumor, thyroid
▪ (-15 to -20C) Muscle, conn tissue, pancreas, uterus, cervix, skin without fat, nonfatty breast tissue, ovary, prostate, tongue, gut
▪ (-35C) Fatty tissue, fatty breast, omental
Advantages:
▪ staining (fat demo by oil red O, silver impregnation, CNS methods)
▪ indispensable for rapid diagnosis during operation
▪ enzyme demo
Disadvantage:
▪ sections don’t form ribbons but stick to the knife blade
(remove with camel’s hair
brush)
▪ lack of embedding mass, distorted structural details during cutting
▪ staining of unfixed tissue is rarely unsatisfactory
▪ produce freezing artifact
STAINING Formation of colors of different tissues and cells -LEEUWENHOEK (saffron) -GOPPERT, COHN (carmine) -GERLACH (sel. Nuclear stain) PURPOSES OF STAINING
▪ render different tissue constituents (more visible)
▪ easier optical differentiation for ID of cells and tissue
▪ display various affinities
▪ display physical char and structural relationships
METHODS TO OBTAIN STAINING REACTION:
▪ Capillary osmosis
▪ Solubility
▪ Adsorption
▪ Absorption
TYPES OR METHODS OF STAINING
Direct Use simple aqueous or alcoholic solution of the dye
Indirect ▪Mordant (link/bridge to form color)
▪Accentuator (accelerate/hasten the speed of staining)
Progressive Not washed or decolorized
▪ less favorable than regressive. Difficultly of producing
intense cell structure, diffused and obscured effect
Regressive Gram staining excess stain is decolorized
▪differentiation/decolorization: selective removal of excess
stain from tissue
Counterstaining Apply different color/stain to provide contrast and background. C Y T O P L A S M I C
▪RED ▪Yellow ▪Green
-Eosin Y,B -Picric acid -Light green SF -Phloxine B -Orange G -Lissamine green -Rose Bengal N U C L E A R
▪RED ▪Blue
-Neutral red -Methylene blue -Safranin red -Toluidine blue -carmine -celestine blue -hematoxylin
Metachromatic Use specific dye (metachromasia) Thiazine and triphenylmethane group -methyl violet/crystal violet -cresyl blue -safranin -bismarck brown -basic fuchsin -methylene blue -thionine -toluidine blue -Azure A,B,C
Microanatomical Demostrante general relationship of tissue and cell without emphasizing the inclusion bodies
▪Cytoplasmic staining
-doesn’t differentiate tissue structure in general
▪Negative staining
-demonstrate bacterial morphology in black background (india ink)
Metallic Impregnation
Demonstrate tissue elements not by stains but by colorless solutions of metallic salts. *adsorption The most valuable metal: gold (gold chloride) and silver (silver nitrate)
▪ Explosive
▪ avoid silver glasswares
▪ don’t expose to sunlight
▪avoid metallic instruments
Vital SUPRAVITAL: -neutral red -janus green -tryphan blue -nile blue -thionine -toluidine blue
▪ Selective staining of living cell constituents
▪demonstrate cytoplasmic structures by phagocytosis of
dye particle
▪nucleus is resistant to vital stain
INTRAVITAL STAINING inject the dye into any part of animal body (intravenous/intraperitoneal/subcutaneous) SUPRAVITAL STAINING stain living cells immediately
COMPOSITION OF DYES
NA
TUR
AL
Fro
m p
lan
ts, a
nim
al
(fo
r w
oo
l & c
ott
on
)
Hematoxylin ▪ Low affinity for tissue, must use mordants (alum, iron, chromium,
copper salts)
▪ Extraction from
heartwood of Mexican Tree: Hematoxylin
campechianum
▪ Hematin
Active coloring agent, oxidized
hematoxylin
▪ Ripening
Oxidation process of hematoxylin to
hematin. (exposure of stain to
air and sunlight) OXA: -H2O2 -HgCl2
-KMnO4 -Na perborate
-NaI
Alum Hematoxylin ▪ For progressive
staining
▪ Counterstained with congo red & safranin.
▪ Salt lakes color: Blue
▪ Blueing: process of
passing the tissue to alkaline solution to
neutralize acid.
▪ Cold water (slows down)
▪ warm water (accelerates)
▪ Very cold water (<10C, produce pink
artifacts)
a) Ehrlich’s hematoxylin
▪ Regressive staining
▪ for muco polysaccharide, cartilage, cement lines of bones b) Harris Hematoxylin
▪ for routine nuclear staining
▪ for exfoliative cytology
▪ for staining sex chromosomes
c) Cole’s hematoxylin
▪ for routine purposes
▪ used in sequence with Celestine blue
d) Mayer’s hematoxylin
▪ used in Celestine blue
▪ for nuclear staining
Iron Hematoxylin ▪ Mordants: ferric ammonium sulfide
(iron alum)
a) Weigert’s hematoxylin
▪ std. for lab
▪ muscle fiber demo
▪ conn.tissue demo
b) Heidenhain’s hematoxylin
▪ cytological stain
▪ for regressive staining of thin sections
▪ for nuclear and cytoplasmic
inclusion (chromatin, chromosome, nucleoli, centrosome, mitochondria) c) Phospho tungstic acid hematoxylin (PTAH)
▪ for structures in paraffin, celloidin, frozen section
Cochineal Dyes ▪ old histologic dye
▪ from extraction of female cochineal bug (coccus cacti)
▪ treated with alum to produce carmine dye
▪ combination with
picric acid/ picrocarmine is used for neutropathological study
▪ combination with
AlCl3/ best’s carmine is used for glycogen demo
Orcein Dye ▪ vege dyes from
lichens
▪ colorless
▪ combination with ammonia and exposure to air produce blue and violet
Syn
the
tic
dye
s/ c
oal
tar
dye
s /A
nili
ne
dye
s (f
rom
C6H
6 h
ydro
carb
on
be
nze
ne)
▪ Chromophores
-produce visible colors
▪ Chromogens
-benzene compounds with chromophore
▪ Auxochrome
-auxiliary radical (props of electrolytic dissociation) -alter the shades of dye -give the props of forming salts
Dyes: a) Acid Dyes
▪ acid: coloring substance
▪ base: sodium
b) Basic Dyes
▪ acid: sulfuric, acetic or HCl
▪ basic: coloring substance c) Neutral dyes
▪ Acid aq + basic aq
▪ for nucleus and cytoplasm
▪ soluble in alcohol
▪ insoluble in water
DIFFERENT STAINS USED IN HISTOPATHOLOGY
Aniline blue Cytoplasmic, counterstain
epithelial section
Basic fuchsin
Plasma stain acid fast organism, mitochondria, smooth muscle Fuelgen’s + schiff’s reagent: detect aldehydes Van gieson’s : conn.tissue, mucin, elastic tissue
Bismarch brown
Contrast stain gram’s technique, acid fast & papanicolau method, diphtheria organism
Carmine Chromatin stain smear prep… Best carmine solution for glycogen Mucicarmine for mucin
Celestine blue
Resistant to strong acid dyes
for routine staining of fixed sections, +alumH = good nuclear staining
Crystal violet
Nuclear/chromatin stain stain amyloid in frozen section, stain platelet in blood
Eosin Eosin B : bluish deeper red color Eosin Y : yellowish
Stain conn tissue and cytoplasm Routine: Hematoxylin – Eosin – methylene blue
Giemsa stain
Stain blood to differentiate leukocyte
Malachite green
Weakly basic, contrast stain, decolorizer, counterstain
Ascaris eggs, erythrocyte,
Methyl green
Chromatin stain, Gives false positive with mucin secretion
Chromatin green (w/acid)
Methylyne blue
Basic nuclear stain, plasma cells, cytological evaluation
Fresh sputum, malignant cells, bacterial stain, milk grading, diphtheria, nervous tissue,
Methylene violet
Metachromatic dye Leukocytes’s nuclei: reddish purple with methylene blue
Orcein Dermatological study Excellent for elastic fibers
Picric acid Contrast stain Cytoplasmic stain Counterstain Tissue fixative Decalcifying agent
Acid fuchsin, conn.tissue (van gieson) crystal violet
Prussian blue
Colored salt of ferric ferrocyanide Microanatomical color contrast
Manufacture of paints Circulatory system
Toluidine blue
Nuclear stain Substitute Recommended for:
Fixed tissue Thionine in frozen section -nissl granules -chromophilic bodies
COMMONLY USED SPECIFIC TISSUE STAIN
Routine histologic study H & E
Conn. Tissue/ collagen Methylene blue
reticulum Gold method
Elastic tissue Orcein
Muscle tissue Van gieson’s
fibrin H & E
Amyloid Methyl violet
Glycogen Best carmine
Mucin Meyer’s mucicarmine
Acid mucopolysaccharide Toluidine blue
Nissl bodies H & E
Neurons, axons, neurofibrils Bielschowsky’s technique
Myeline sheath Sudan black
Astrocytes Mallory’s PTAH
Argentaffin granules Masson Fontana silver method
Phospholipids Baker’s technique
Neutral fat Sudan black
Fatty acids Lillie’s sulfuric acid nile blue
Hb Amido black
Hemosiderin Prussian blue
Hematoidin Gmelin’s
Bile pigment Gmelin’s
Hemofuchsin pigment Mallory’s fuchsin stain
Melanin Masson Fontana
Bacteria Gram staining
Actinomyces Brown breen’s
Acid fast Ziehl neelsen
Spirochetes Giemsa stain
Fungi Ziehl neelsen
Nocardia Gram staining
Amoeba Heidenhain’s iron hematoxylin
Inclusion bodies/ negri Schleifsten’s method
Calcium Calcium Prussian blue
Urates De galantha stain
ROUTINE HEMATOXYLIN STAINING PROCEDURE
DEPARAFFINIZATION
Xylol I 5 min
Removes paraffin Xylol II
Acetone-alcohol 3 min Removes xylol
Acetone
HYDRATION
Alcohol 95%
3 min Hydrates Alcohol 80%
Alcohol 70%
Alcohol 50%
Wash with distilled water
STAININIG Hematoxylin 5 min Stains nuclei
Rinse with water
DECOLORIZATION Acid alcohol 1 dip Decolorize
BLUEING
Ammonia water
Dip to blue Blues nuclei Alcohol 80%
Alcohol 95%
COUNTERSTAINING Eosin 8 min Stains cytoplasm
DEHYDRATION
Alcohol 95%
3 min Dehydrates and removes excess
stain
Alcohol 95%
Acetone
Acetone-xylol
CLEARING Xylol I
5 min Clears out
alcohol Xylol II
RESULTS
Basophil cytoplasm, plasma cells, osteoblast Purplish
Calcium and calcified bone Purplish blue
Cartilage Pink/light blue to dark blue
Cement line of bones Blue (ehrlich)
Collagen, osteod Light pink
Cytoplasm Pink
Decalcified bone matrix Deep pink
Karyosome Dark blue
Muscle fibers, thyroid colloid, thick elastic fibers Deep pink
Nuclei Blue to blue black
RBC, Eosinophil granules, keratin Bright orange red
DIFFICULTIES IN STAINING
Stains on the skin ▪ poor technique
▪ health hazards
Flake or float off tissue section
▪ dirty or greasy slide
▪ inadequate fixation on the slide
▪ torn section
▪ sections contain air bubbles
▪ inadequate infiltration
▪ albumin adhesive is too old
▪ unthorough spreading
Section doesn’t take up the stain
▪ insufficient/improperly ripened hematoxylin
▪ impurities in the dye/water solvent
▪ inadequate paraffin washing off
▪ loss of staining ability (ppt)
Sections don’t appear clear under microscope
▪ xylol should be replaced
▪ water in absolute alcohol
▪ moisture in the coverslip
▪ too much egg albumin on the slide
▪ decolorizer wasn’t completely removed
RESTAINING OLD SECTIONS (OLD/BLEACHED/FADED)
Xylol (heat until mounting media bubbles) 24 hr
Remove coverslip by dissecting needle
Xylol to remove remaing mounting media 30 min
Wash with water
Potassium permanganate 5-10 min
Wash with water
5% oxalic acid until decolorized 5 min
Wash with water 5 min
Restain
Mount
MOUNTING OF SECTIONS Purpose:
▪ protect spn from physical injury
▪ protect section from bleaching or deterioration
▪ preserve slide for permanent keeping MOUNTING MEDIUM syrupy fluid applied between section and coverslip, preventing movement of the coverslip CHARACTERISTICS OF GOOD MOUNTING MEDIUM
▪ Ref.Index : near to the glass 1.518
▪ shouldn’t dry quickly
▪ shouldn’t dissolve out or fade tissue section
▪ should not cause shrinkage and distortion
▪ should set hard
▪ shouldn’t have granularity/ cracking
▪ should be miscible with xylene or toluene
▪ should be nonreactive, no pH or color change
CLASSIFICATION
Temporary/ Aqueous ▪ for water miscible prep
▪ made of gelatin to solidify medium
▪ made of glycerol to prevent cracking
▪ made of sugar to increase ref. index
▪ made of a prsv. Solution
Permanent/ Resinous ▪ for dehydrated prep and cleared prep
▪ natural/synthetic
PERMANENT MOUNTING MEDIA
Canada balsam ▪ from Canadian tree (abus balsamea)
▪ dissolved in xylene
▪ recommended for whole mounts and thick section
▪ darken slightly with age
DPX/ Kirkpatrick and lendrum
▪Ref. index = 1.52
▪ recommended for small tissue section
Clarite X ▪ most widely used in north America
▪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
S E A L I N G
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
SPECIAL PROCESSING TECHNIQUE
when chemical constituents of tissues shouldn’t have been altered/removed/
displaced Frozen Section most ideal tissue prsv. To avoid complete or partial loss of enzyme General Principle
▪ Quenching/freezing = produce instant cessation of cellular activities, prevent
chemical alteriation
▪ rapid freezing = prevent ice crystal artifacts formation
▪ freezing agents: Liquid nitrogen
▪ isopentane, pentane, propane = cooled to very low temp, to retain fluidity
METHODS TO AVOID CHEMICA FIXATION OF BLOCKS
FREEZE DRYING -quenching/rapid freezing -dessication (removal of water) DISADVANTAGE -time consuming -expensive -diff.to section -brittle -not for routine
▪ 2mm tissue + freezing agent + liquid nitrogen
▪ solidification 2-3sec = prevent ice crystal artifacts
▪ high vacuum drying apparatus
▪ water sublimation
▪ dessication 24-48hr
▪ infiltration, impregnation in vacuum embedding oven
▪ sectioning
▪ staining
ADVANTAGES: -min. shrinkage -fresh tissue -min. chemical change -less displacement -for enzyme studies
FREEZE SUBSTITUTION -rapid freezing -subsequent infiltration, embedding
▪ fixation with rossman’s fluid or osmium tetroxide in 1%
acetone 4-6 days (-60 to -70C)
▪ infiltration, embedding
▪ more economical
▪ recommended for routine
FRESH FROZEN TISSUE SECTIONING
▪ maintain fresh and solid state
DECALCIFICATION Process of removing calcium salts or lime salts and its deposits from hard tissue for softening of tissue to facilitate embedding and sectioning. FIXATION DECALCIFICATION IMPREGNATION **Inadequate decalcification = poor cutting of hard tissue, damage to knife edge Tissues for decalcification: -bones -teeth -atheromatous aorta -calcified tuberculous foi BEFORE CALCIFICATION
▪ 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 used
▪ stable
▪ easily available
▪ relatively
inexpensive
The rate is affected by:
▪ tissue structure
▪ temp
▪ volume and conc.
Of solution
▪ external factor
(accelerate)
Nitric acid HCl Formic acid Trichloroacetic acid Sulfurous acid Chromic acid Citric acid
Chelating agents
Combines Ca2+ to form soluble non ionized complex, facilitate removal of calcium.
Advantage: -forms min. histological artifacts Disadvantage: -slow reacting
▪ EDTA/
Versene
Ion exchange resin
Ammonium form of plystyrene resin
▪ remove calcium
ions from formic acid , increase solubility
Advantages: Cellular detail is well preserved
Electrical ioinization/ electrophoresis
▪ Ca2+ are
attracted to negative electrode, then removed from decalcifying solution.
Advantage:
▪Faster due to heat and electrolyte reaction
▪ recommended for
small bone fragments Disadvantage: Not well preserved
NITRIC ACID: most common, very rapid, min. distortion, inhibits nuclear staining, destroy tissue
Aquous nitric acid solution 10%
12-24 hr ▪ rapid
▪ for urgent biopsy
▪ prolonged may
distort tissue Formol nitric acid 1-3 days
Perenyi’s fluid 2-7 days ▪ no maceration ▪ slow
Phloroglucin nitric acid
12-24 hr ▪ most rapid ▪ poor nuclear
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 sodium citrate
3-14 days
▪ excellent nuclear staining
▪ slow
▪ 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
WAYS TO DETERMINE THE EXTENET OF DECALCIFICATION
Physical/mechanical test ▪Touching/bending tissue with fingers
▪determine consistency
X-RAY or radiological method ▪can detect smallest focus of Ca
▪ very expensive
Chemical method ▪ simple, reliable, convenient
CLOUDY: -Incomplete -presence of Ca NOT CLOUDY: -complete -no more Ca