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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 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 - For paraffin-embedded sections - most common type used for routine & research laboratories - 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 - 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 - For electron microscopy

Belgium yellow/ hone Arkansas Fine carborundum Plate glass hone Machine hone

Sliding (celloidinembedded section)

TYPES OF HONES for manual sharpening usually gives best result gives more polishing effect much coarser used for badly nicked knives use diamanthine for final polishing glass disc or wheel driven by electric motor

Rotary Freezing

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 shouldnt 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 Egg white + glycerin + thymol crystal Mayers albumin Dried albumin Dried albumin + NaCl + thymol crystal Gelatin Gelatin + water + glycerol +phenol crystal Gelatin chrome Can be added to water bath. alum 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 REASON REMEDY
prolonged fixation prolonged dehydration prolonged clearing prolonged paraffin infiltration in over heated paraffin oven drying out of tissue before fixation water not removed completely (incomplete dehydration) incomplete removal of clearing agent due to insufficient impregnation Soften tissue by soaking oil in a smaller dish or bowl (w/water) with detergent, phenol or molliflex


Ultra thin sectioning microtome

Block Holder/ chuck Knife carrier and knife Pawl, ratchet feed wheel, adjs.screw

MAJOR PARTS OF A MICROTOME Holds the tissue block in place Actual cutting of tissues 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 Cutting edge PARTS OF MICROTOME KNIFE Cutting facet, made of good quality steel. Able to cut good section from a paraffin wax block without any serration note on examination. Spring-loaded semicircular sheet of metal slipped on the knife Angle between the cutting edge: 27-32 Angle formed by the sides of the wedge knife (5-14) Angle between cutting facet (5-15) KINDS OF MICROTOME KNIVES 250mm, 1 side is flat, 1 side is concave 120mm, both sides concave 100mm, both sides straight CARE OF MICROTOME KNIFE removal of gross nicks on the knife edge (coarse hoaning) removal of blemishes and irregularities from knife edge Direction: heel to toe for sharpening removal of burr or irregularities during honing final polishing of the knife edge Direction: toe to heel (reverse of honing)

Wedge Knife back Bevel angle Wedge angle Clearance angle


Plane concave Biconcave Plane wedge

Brittle/hard tissue


Clearing becomes milky as soon as tissue is placed in it On trimming, tissue smells of clearing agent Tissue is opaque section cutting is difficult due to presence of alcohol Tissue shrinks away from the wax when

Repeat dehydration with absolute alcohol, then clear again Trim the blocks down nearest to the tissue Melt the remaining wax on embedding oven Repeat paraffin impregnation Repeat clearing


insufficient clearing

STROPPING Hone: a natural stone or hard grinding surface for sharpening a knife

insufficient dehydration

Repeat whole procedure

trimmed Tissue is soft when block is trimmed Airholes found on tissue during trimming (appear crystalline) Moist & crumbled Paraffin block after cooling

incomplete fixation incomplete impregnation contaminated wax block not cooled rapid enough insufficient paraffin impregnation 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 knife is dull knife is tilted too much knife edge is dirty blunt or dull spot on knife (irregular knife edge)

Repeat fixation Reembed in freshly filtered wax

Resistance is felt on the lower part of section during cutting Horizontal or parallel lines or forrows across the sections/chatters are seen, forming thin and thick zones

Repeat paraffin impregnation, reembed 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

tilt of knife is insufficient, paraffin block is therefore compressed against the base of knife towards the end of the stroke knife edge vibrates due to: a) hardness of the tissue b) tilt of the knife is too great knife is blunt knife isnt 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 isnt cut tilt of knife is too slant or too big tissue is too hard knife blade is too thin freezing isnt adequate tissue is frozen too hard

increase the tilt

treat with phenol during processing or collodionize

Section cut is sometimes thin, sometimes thick

tighten adjusting and locking screws increase tilt

Sections fail to form ribbons

Sections roll up on cutting so that they adhere and get broken and against the knife edge

Sharpen the knife Reduce the tilt Clean the knife edge

Ribbon is curved, crooked or uneven instead of straight

Sections are compressed, wrinkled or jammed

Sections are torn and crumble when cut

edges of the block are not parallel but round or wedgeshaped knife is not parallel to the block paraffin is impure 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 incomplete dehydration, clearing and infiltration of tissue with wax paraffin is warm and soft knife is blunt

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

Knife makes a hard metallic scraping or ringing sound on backstroke, when sectioning Frozen tissue crumbles and comes off the blockholder when cut Frozen tissue chips into fragments when cut

readjust the angulation of the knife take fresh block change the knife Refreeze the tissue

warm the tissue with fingers

DIFFICULTIES DERIVED FROM THE TISSUES blood clot, cervix, thyroid tissue fatty tissues (subcutaneous tissue, breast, lipoma) brain and lymph nodes soft tissue
very hard in routine processing soft blocks and shredded tissue block before cutting, effect with a firm sharp stroke. Use chloroform as clearing agent. Use tetrahydrofuran for dehydrating agent. Cut thin 2mm, and impregnate with wax in vacuum bath

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 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 to hour. sharpen the knife resharpen, using a knife back or automatic knife sharpener reembed in freshly filtered wax if necessary once embedded in paraffin wax, decalcification is impractical use a base sledge microtome with a wedge knife reduce the tilt

very hard and brittle (if using xylol for dealcoholization) Tend to expand more when floated out

Use chloroform and vacuum impregnation with lymph nodes 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 dont 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 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 CO2 (Freezing agent) different temp between knife and tissue (latter is colder) Advantages: staining (fat demo by oil red O, silver impregnation, CNS methods) indispensable for rapid diagnosis during operation enzyme demo 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. Disadvantage: sections dont form ribbons but stick to the knife blade (remove with camels hair brush) lack of embedding mass, distorted structural details during cutting staining of unfixed tissue is rarely unsatisfactory produce freezing artifact

Sections are squashed width of each section less than that of the block

Bevel of knife is lost due to incorrect sharpening bubble or dirt formed in the embedding medium hard spot in tissue possibly due to calcium

A Hole is formed in the section


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 static electricity due to low atmospheric humidity knife edge is dirty knife edge is dull knife tilt is too great nicks/damage on the knife edge dirty embedding medium knife edge is dirty tilt of the knife is too great knife tilt is too great knife is dull paraffin is too soft r RT is warm

tighten screw cut blocks into smaller fragments soften blocks in detergent or phenol 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 sharpen the knife reembed in filetered wax clean the knife edge with xylol cool paraffin wax in ice water

Sections adhere to the knife or other parts of the machine

Ribbon is split or lengthwise vertical scratches are seen on section Sections are lifted from the knife on upstrokes

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 Use simple aqueous or alcoholic solution of the dye Mordant (link/bridge to form color) Accentuator (accelerate/hasten the speed of staining) Not washed or decolorized less favorable than regressive. Difficultly of producing intense cell structure, diffused and obscured effect Gram staining excess stain is decolorized differentiation/decolorization: selective removal of excess stain from tissue Apply different color/stain to provide contrast and background. CYTOPLASMIC RED Yellow Green -Eosin Y,B -Picric acid -Light green SF -Phloxine B -Orange G -Lissamine green -Rose Bengal NUCLEAR RED Blue -Neutral red -Methylene blue -Safranin red -Toluidine blue -carmine -celestine blue -hematoxylin 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 Demostrante general relationship of tissue and cell without emphasizing the inclusion bodies Cytoplasmic staining -doesnt differentiate tissue structure in general Negative staining -demonstrate bacterial morphology in black background (india ink) 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 dont expose to sunlight avoid metallic instruments 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


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) Ehrlichs 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) Coles hematoxylin for routine purposes used in sequence with Celestine blue d) Mayers hematoxylin used in Celestine blue for nuclear staining a) Weigerts hematoxylin std. for lab muscle fiber demo conn.tissue demo b) Heidenhains 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

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

Direct Indirect Progressive

Iron Hematoxylin
Mordants: ferric ammonium sulfide (iron alum)



NATURAL From plants, animal (for wool & cotton)

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/ bests carmine is used for glycogen demo


Orcein Dye
vege dyes from lichens colorless combination with ammonia and exposure to air produce blue and violet


Metallic Impregnation

Vital SUPRAVITAL: -neutral red -janus green -tryphan blue -nile blue -thionine -toluidine blue

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

Synthetic dyes/ coal tar dyes /Aniline dyes (from C6H6 hydrocarbon benzene)

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, epithelial section counterstain Basic Plasma stain acid fast organism, mitochondria, fuchsin smooth muscle Fuelgens + schiffs reagent: detect aldehydes Van giesons : conn.tissue, mucin, elastic tissue Bismarch Contrast stain grams technique, acid fast & brown papanicolau method, diphtheria organism Carmine Chromatin stain smear prep Best carmine solution for glycogen Mucicarmine for mucin Celestine Resistant to strong acid for routine staining of fixed sections, blue dyes +alumH = good nuclear staining Crystal Nuclear/chromatin stain stain amyloid in frozen section, stain violet platelet in blood Eosin Eosin B : bluish deeper Stain conn tissue and cytoplasm red color Routine: Hematoxylin Eosin Eosin Y : yellowish methylene blue Giemsa stain Malachite green Methyl green Methylyne blue Methylene violet Orcein Picric acid Stain blood to differentiate leukocyte Ascaris eggs, erythrocyte,

Fungi Nocardia Amoeba Inclusion bodies/ negri Calcium Urates

Ziehl neelsen Gram staining Heidenhains iron hematoxylin Schleifstens method Calcium Prussian blue De galantha stain

Weakly basic, contrast stain, decolorizer, counterstain Chromatin stain, Gives false positive with mucin secretion Basic nuclear stain, plasma cells, cytological evaluation Metachromatic dye Dermatological study Contrast stain Cytoplasmic stain Counterstain Tissue fixative Decalcifying agent Colored salt of ferric ferrocyanide Microanatomical color contrast Nuclear stain Substitute Recommended for:

Chromatin green (w/acid)

ROUTINE HEMATOXYLIN STAINING PROCEDURE Xylol I Removes 5 min paraffin Xylol II DEPARAFFINIZATION Acetone-alcohol 3 min Removes xylol Acetone Alcohol 95% Alcohol 80% HYDRATION 3 min Hydrates Alcohol 70% Alcohol 50% Wash with distilled water STAININIG Hematoxylin 5 min Stains nuclei Rinse with water DECOLORIZATION Acid alcohol 1 dip Decolorize Ammonia water BLUEING Alcohol 80% Dip to blue Blues nuclei Alcohol 95% COUNTERSTAINING Eosin 8 min Stains cytoplasm Alcohol 95% Dehydrates and Alcohol 95% DEHYDRATION 3 min removes excess Acetone stain Acetone-xylol Xylol I Clears out CLEARING 5 min alcohol Xylol II RESULTS Basophil cytoplasm, plasma cells, osteoblast Calcium and calcified bone Cartilage Cement line of bones Collagen, osteod Cytoplasm Decalcified bone matrix Karyosome Muscle fibers, thyroid colloid, thick elastic fibers Nuclei RBC, Eosinophil granules, keratin

Fresh sputum, malignant cells, bacterial stain, milk grading, diphtheria, nervous tissue, Leukocytess nuclei: reddish purple with methylene blue Excellent for elastic fibers Acid fuchsin, conn.tissue (van gieson) crystal violet

Prussian blue

Manufacture of paints Circulatory system Fixed tissue Thionine in frozen section -nissl granules -chromophilic bodies

Purplish Purplish blue Pink/light blue to dark blue Blue (ehrlich) Light pink Pink Deep pink Dark blue Deep pink Blue to blue black Bright orange red

Toluidine blue

COMMONLY USED SPECIFIC TISSUE STAIN Routine histologic study H&E Conn. Tissue/ collagen Methylene blue reticulum Gold method Elastic tissue Orcein Muscle tissue Van giesons fibrin H&E Amyloid Methyl violet Glycogen Best carmine Mucin Meyers mucicarmine Acid mucopolysaccharide Toluidine blue Nissl bodies H&E Neurons, axons, neurofibrils Bielschowskys technique Myeline sheath Sudan black Astrocytes Mallorys PTAH Argentaffin granules Masson Fontana silver method Phospholipids Bakers technique Neutral fat Sudan black Fatty acids Lillies sulfuric acid nile blue Hb Amido black Hemosiderin Prussian blue Hematoidin Gmelins Bile pigment Gmelins Hemofuchsin pigment Mallorys fuchsin stain Melanin Masson Fontana Bacteria Gram staining Actinomyces Brown breens Acid fast Ziehl neelsen Spirochetes Giemsa stain

DIFFICULTIES IN STAINING poor technique health hazards Flake or float off tissue dirty or greasy slide section inadequate fixation on the slide torn section sections contain air bubbles inadequate infiltration albumin adhesive is too old unthorough spreading Section doesnt take up the insufficient/improperly ripened hematoxylin stain impurities in the dye/water solvent inadequate paraffin washing off loss of staining ability (ppt) Sections dont appear clear xylol should be replaced under microscope water in absolute alcohol moisture in the coverslip too much egg albumin on the slide decolorizer wasnt completely removed Stains on the skin 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 shouldnt dry quickly shouldnt dissolve out or fade tissue section should not cause shrinkage and distortion should set hard shouldnt have granularity/ cracking should be miscible with xylene or toluene should be nonreactive, no pH or color change CLASSIFICATION 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 for dehydrated prep and cleared prep natural/synthetic PERMANENT MOUNTING MEDIA from Canadian tree (abus balsamea) dissolved in xylene recommended for whole mounts and thick section darken slightly with age Ref. index = 1.52 recommended for small tissue section most widely used in north America Synthetic resin ref index = 1.544 synthetic resin mixture in xylene pale yellow/colorless dries quickly without retraction, prsv. Stains well

SPECIAL PROCESSING TECHNIQUE when chemical constituents of tissues shouldnt 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 2mm tissue + freezing agent + liquid nitrogen -quenching/rapid solidification 2-3sec = prevent ice crystal artifacts freezing high vacuum drying apparatus -dessication water sublimation (removal of water) dessication 24-48hr infiltration, impregnation in vacuum embedding oven sectioning staining DISADVANTAGE -time consuming -expensive -diff.to section -brittle -not for routine FREEZE SUBSTITUTION -rapid freezing -subsequent infiltration, embedding FRESH FROZEN TISSUE SECTIONING ADVANTAGES: -min. shrinkage -fresh tissue -min. chemical change -less displacement -for enzyme studies fixation with rossmans fluid or osmium tetroxide in 1% acetone 4-6 days (-60 to -70C) infiltration, embedding more economical recommended for routine maintain fresh and solid state

Temporary/ Aqueous

Permanent/ Resinous

Canada balsam

DPX/ Kirkpatrick and lendrum Clarite X


SEMIPERMANENT OR TEMPORARY MOUNTING MEDIA 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 apathys gum syrup for methylene blue stained nerve prep gen.purpose aqueous mountant ref index 1.52 Bruns fluid recommended for mounting frozen sections from water Levulose/ fructose high ref index 1.5 doesnt leach metachromatic stain Mineral oil/ liquid paraffin for romanowsky stained smear Water SEALING RINGING process of sealing the margins of coverslip to prevent escape of fluid. Ringing media: -paraffin wax -kronigs/Dunoyers 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 / 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 hellys fluid 15-24hr GOOD DECALCIFYING AGENT: can remove calcium salts completely doesnt produce destruction of cells DECALCIFYING AGENTS The rate is affected most widely by: used tissue structure stable temp easily available volume and conc. relatively Of solution inexpensive external factor (accelerate) Combines Ca2+ to form soluble non ionized complex, facilitate removal of calcium. Ammonium form of plystyrene resin remove calcium ions from formic acid , increase solubility Ca2+ are attracted to negative electrode, then removed from decalcifying solution. Advantage: -forms min. histological artifacts Disadvantage: -slow reacting Advantages: Cellular detail is well preserved

WAYS TO DETERMINE THE EXTENET OF DECALCIFICATION Physical/mechanical test Touching/bending tissue with fingers X-RAY or radiological method Chemical method determine consistency can detect smallest focus of Ca very expensive simple, reliable, convenient CLOUDY: -Incomplete -presence of Ca NOT CLOUDY: -complete -no more Ca


Nitric acid HCl Formic acid Trichloroacetic acid Sulfurous acid Chromic acid Citric acid EDTA/ Versene

Chelating agents

Ion exchange resin

Electrical ioinization/ electrophoresis

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 12-24 hr rapid prolonged may solution 10% distort tissue for urgent biopsy Formol nitric acid 1-3 days 2-7 days Perenyis fluid 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 slow Formic acid 3-14 excellent nuclear 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/FLEMMINGS FLUID: fixative + decalcifying agent, inhibit nuclear staining CITRIC ACID-CITRATE BUFFER 4.5pH: use chloroform as prsv., excellent nuclear and cytoplasmic staining VON EBNERS FLUID: good cytologic staining, for teeth and small bones DEGREE OF DECALCIFICATION: shortened/prolonged = unsatisfactory prolonged = maceration, destruction underdecalcification = interfere normal cutting