SECTIONING 徐智慧/
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
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 徐智慧/ ▪ 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 徐智慧/ 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 TU R A L Fr o m pl ant s, a ni m al (for wool & cot ton) 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 Sy nt h e ti c dye s/ coal t ar d ye s /A ni line dye s (from C6 H6 hy d rocar bon b e nz e 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 徐智慧/ 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
DECALCIFICATION 徐智慧/
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
