Theories of staining (2) / dental implant courses by Indian dental academy

TTHHEEOORRIIEESS OOFF SSTTAAIINNIINNGG
INDIAN DENTAL ACADEMYINDIAN DENTAL ACADEMY
Leader in continuing Dental EducationLeader in continuing Dental Education
www.indiandentalacademy.comwww.indiandentalacademy.com

ReferencesReferences
1.1. Cellular Pathology TechniqueCellular Pathology Technique
C.F.A CullingC.F.A Culling
R.T AllisonR.T Allison
W.T BarrW.T Barr
2.2. Theory and Practice of Histological TechniqueTheory and Practice of Histological Technique
John D BancroftJohn D Bancroft
3.3. Textbook of Oral PathologyTextbook of Oral Pathology
Anil GhomAnil Ghom
Shubhangi MhaskeShubhangi Mhaske

IntroductionIntroduction
 StainingStaining
 C.F.A Culling et al.C.F.A Culling et al.
 ““ Treating tissue or cells with a reagent or series ofTreating tissue or cells with a reagent or series of
reagents so that it acquires a colour ; usually noreagents so that it acquires a colour ; usually no
particles of dye are seen and the stained element isparticles of dye are seen and the stained element is
transparent”transparent”
 BancroftBancroft
 ““ Visual labeling of some entity by attaching orVisual labeling of some entity by attaching or
depositing in its vicinity, a marker of characteristic colourdepositing in its vicinity, a marker of characteristic colour
or shape.or shape.

 Biological stainingBiological staining : “ Is the union between a coloured: “ Is the union between a coloured
dye and a tissue substrate which resists simpledye and a tissue substrate which resists simple
washing”washing”
 Stain is any substance which when added to living cellsStain is any substance which when added to living cells
or to fixed structures or structural components makesor to fixed structures or structural components makes
them clearly visible or detectable.them clearly visible or detectable.

CHARACTERISTIC FEATURES OF AN IDEAL STAIN:CHARACTERISTIC FEATURES OF AN IDEAL STAIN:
 An ideal stain should allow for:An ideal stain should allow for:
 Evaluation of architectural patterns of the tissue fragments.Evaluation of architectural patterns of the tissue fragments.
 Proper evaluation of nuclear morphology; i.e all the nuclearProper evaluation of nuclear morphology; i.e all the nuclear
parameters should be clearly visualized.parameters should be clearly visualized.
 Details of cytoplasmic characteristics.Details of cytoplasmic characteristics.
 Visualization and identification of diagnostic features in theVisualization and identification of diagnostic features in the
background.background.

 Staining agent =chromogen + auxochromeStaining agent =chromogen + auxochrome
 Chromogen- colouring agentChromogen- colouring agent
 Auxachrome- stains tissue & make itAuxachrome- stains tissue & make it
resistant to washing.resistant to washing.
 Chromogens - azo, nitro, nitroso, quinoid,Chromogens - azo, nitro, nitroso, quinoid,
ethylene group.ethylene group.
 Auxochromes - sulfates , carboxyls,Auxochromes - sulfates , carboxyls,
hydroxylshydroxyls
 Eg. – picric acid = nitro+ hydroxyEg. – picric acid = nitro+ hydroxy

 Biological stains have been used to visualize and identifyBiological stains have been used to visualize and identify
tissue and cell components .tissue and cell components .
 Coloring or dyeing or staining of tissues :Coloring or dyeing or staining of tissues :
 Makes it possible to see and study the physical featuresMakes it possible to see and study the physical features
andand
 To study the relationship of tissues and their constituentTo study the relationship of tissues and their constituent
cells.cells.

Successful stainingSuccessful staining
 SpecificitySpecificity – is ability to distinguish between the– is ability to distinguish between the
individual cell componentsindividual cell components
 SensitivitySensitivity – capacity of a stain to demonstrate tissue– capacity of a stain to demonstrate tissue
substance at low concentrationsubstance at low concentration
Satisfactory staining method…Satisfactory staining method…
high sensitivity + high specificityhigh sensitivity + high specificity

Affinity of stains to tissueAffinity of stains to tissue
 Depends on 2 factorsDepends on 2 factors
 PHYSICAL FACTORSPHYSICAL FACTORS
 OsmosisOsmosis
 AbsorptionAbsorption
 Selective adsorptionSelective adsorption
 CHEMICAL FACTORSCHEMICAL FACTORS
 Based on acidic and basic nature of tissueBased on acidic and basic nature of tissue
Acidic tissues have affinity for basic stainAcidic tissues have affinity for basic stain
Basic tissues have affinity for acidic stainBasic tissues have affinity for acidic stain

ImpregnationImpregnation
 Unlike staining, makes use of salts of heavy metalsUnlike staining, makes use of salts of heavy metals
which are precipitated selectively on cellular and tissuewhich are precipitated selectively on cellular and tissue
componentscomponents
 Used commonly to demonstrateUsed commonly to demonstrate
 Reticulin fibres Minute organisms likeReticulin fibres Minute organisms like
spirochaetesspirochaetes
Commonly used compound –Commonly used compound – silver nitratesilver nitrate

Basic structure of dyesBasic structure of dyes
 2 basic types2 basic types
 NaturalNatural
 derived from plants and animalsderived from plants and animals
 SyntheticSynthetic
 derived from coal gas industryderived from coal gas industry
 Have aromatic hydrocarbon benzene as their centralHave aromatic hydrocarbon benzene as their central
componentcomponent
 Benzene has a ring structureBenzene has a ring structure
which gives flexibilitywhich gives flexibility
(double bonds are not static)(double bonds are not static)

BenzeneBenzene
 Benzene is not itself colouredBenzene is not itself coloured
 Chemical group when introduced into benzene ring,Chemical group when introduced into benzene ring,
induces colour to compound.induces colour to compound.
 These groupings –These groupings – chromophoreschromophores
 Resultant structure -Resultant structure - chromogenchromogen

AuxochromesAuxochromes
 Chromogen – not a dyeChromogen – not a dye
 Consist ofConsist of
 organic compound with chromophoreorganic compound with chromophore
 Ionising radicle (Auxochrome)Ionising radicle (Auxochrome)
 This auxochrome acts as an electron donor (-OH, -NH2,This auxochrome acts as an electron donor (-OH, -NH2,
-SO2H, -COOH)-SO2H, -COOH)

ChromophoresChromophores
 3 types3 types
 Nitro groupingNitro grouping
 -NO2-NO2
 Dyes include picric acid and AuramineDyes include picric acid and Auramine
 Azo couplingAzo coupling
 N=NN=N
 Dyes include orange G and congo redDyes include orange G and congo red

Quinonoid groupQuinonoid group
 -C=C--C=C-
 Dyes include basic and acid fuchsin, eosin, crystal violet,Dyes include basic and acid fuchsin, eosin, crystal violet,
methylene blue, neutral red and natural dyes (haematin)methylene blue, neutral red and natural dyes (haematin)
 Aryl methane eg Cystal VoiletAryl methane eg Cystal Voilet
 Xanthene eg EosinXanthene eg Eosin
 Quinoline eg Thioflavin TQuinoline eg Thioflavin T
 Thiazol eg Neutral Red ,SafraninThiazol eg Neutral Red ,Safranin
 Quinoneimine eg Celestine blue, Methylene blueQuinoneimine eg Celestine blue, Methylene blue
 Anthraquinone eg Alcian blueAnthraquinone eg Alcian blue

Classification of dyesClassification of dyes
 Based on originBased on origin
 NaturalNatural
 SyntheticSynthetic
 Based on the physicochemical properties of the dyeBased on the physicochemical properties of the dye
 Flourescent (Thioflavin)Flourescent (Thioflavin)
 Metachromatic (Toulidine blue)Metachromatic (Toulidine blue)
 Leuco (Leuco Methylene blue)Leuco (Leuco Methylene blue)
 Neutral (azure-eosinate)Neutral (azure-eosinate)

 Based on the dye structureBased on the dye structure
 Metal complex (aluminium or iron complexes of hematin)Metal complex (aluminium or iron complexes of hematin)
 Azo (orange G )Azo (orange G )
 Xanthene (pyronin Y)Xanthene (pyronin Y)
 Based on dye usage in biological stainingBased on dye usage in biological staining
 Fluorescent probe (janus green)Fluorescent probe (janus green)
 Fat (oil red)Fat (oil red)
 Mucin (alcian blue)Mucin (alcian blue)

 Based on dye usage in textile industryBased on dye usage in textile industry
 Acid (eosin)Acid (eosin)
 Basic (safranin)Basic (safranin)
 Neutral (romanowsky stain)Neutral (romanowsky stain)
 Based on the mode of action of dyeBased on the mode of action of dye
 Mordant (gallocyanin chrome alum)Mordant (gallocyanin chrome alum)
 Reactive (mercury orange)Reactive (mercury orange)

Staining properties of dyesStaining properties of dyes
 2 types2 types
 MicroanatomicalMicroanatomical
 Demonstrates relationship of tissue to each other. clearlyDemonstrates relationship of tissue to each other. clearly
differentiates nuclei from cytoplasmdifferentiates nuclei from cytoplasm
 CytologicalCytological
 Demonstrates tiny structures in nucleus and cytoplasmDemonstrates tiny structures in nucleus and cytoplasm
and any of the tissue typesand any of the tissue types

Types of stainingTypes of staining
 Indirect stainingIndirect staining
 When a mordant is applied (H and E Method)When a mordant is applied (H and E Method)
 Direct stainingDirect staining
 When a mordant is not necessary (alcoholic or aniline stains)When a mordant is not necessary (alcoholic or aniline stains)
 Progressive stainingProgressive staining
 Stains which colour tissue in a definite order (mayer sStains which colour tissue in a definite order (mayer s
hematoxylin for nuclei)hematoxylin for nuclei)
 Regressive stainingRegressive staining
 Involves overstaining of all tissue structuresInvolves overstaining of all tissue structureswww.indiandentalacademy.comwww.indiandentalacademy.com

 Vital stainingVital staining
 When inclusions of live cells or tissues are stainedWhen inclusions of live cells or tissues are stained
 Supravital stainingSupravital staining
 When living cells are stained after being removed from the bodyWhen living cells are stained after being removed from the body
 Intravital stainingIntravital staining
 Staining of cells while still part of the bodyStaining of cells while still part of the body

 Negative stainingNegative staining
 To examine bacterial morphology and capsuleTo examine bacterial morphology and capsule
 Metachromatic stainingMetachromatic staining
 Depends on the dye and nature of tissue ( toulidine blue )Depends on the dye and nature of tissue ( toulidine blue )

Production of colourProduction of colour
Double valency bonds of benzene are not stableDouble valency bonds of benzene are not stable
Light waves --- excite electronsLight waves --- excite electrons
Change in bond position (resonance)Change in bond position (resonance)
Excitement of benzene absorption bands pushed to higherExcitement of benzene absorption bands pushed to higher
wavelenghtswavelenghts
Colour visible to the eyeColour visible to the eye

Commonly used terminologiesCommonly used terminologies
 MordantMordant
 Metallic substances – acts as a link between stain andMetallic substances – acts as a link between stain and
tissuetissue
 Mordant + dye ChemicalMordant + dye Chemical LAKELAKE
combinationcombination
 Lake is invariably basic in actionLake is invariably basic in action
 AdvantageAdvantage of dye-mordant-tissue complexof dye-mordant-tissue complex
 Insoluble in most fluids used in biological stainingInsoluble in most fluids used in biological staining
 Little altered by treatment of tissue sectionLittle altered by treatment of tissue section

MordantsMordants
 Mordants are used in 3 waysMordants are used in 3 ways
 BeforeBefore the application of stain (premordanting)the application of stain (premordanting)
 Eg: Heidenhain s iron hematoxylinEg: Heidenhain s iron hematoxylin
 InIn conjunctionconjunction with stain (metachromatic staining)with stain (metachromatic staining)
 Eg: Ehrlich s acid alum hematoxylinEg: Ehrlich s acid alum hematoxylin
 AfterAfter application of stain (post mordanting)application of stain (post mordanting)
 Eg: gram stainEg: gram stain

Commonly used terminologiesCommonly used terminologies
 AccentuatorsAccentuators
 Chemical substances which increase theChemical substances which increase the
 Colouring powerColouring power
 crispnesscrispness
 Selectivity of stainSelectivity of stain
 Also known asAlso known as acceleratorsaccelerators
 Eg: phenol in carbol fuschinEg: phenol in carbol fuschin
 Aniline in gentian violetAniline in gentian violet

Staining properStaining proper

 Staining involves the visual labeling of some entity byStaining involves the visual labeling of some entity by
attaching or depositing in its vicinity, a marker ofattaching or depositing in its vicinity, a marker of
characteristic colour and shape.characteristic colour and shape.
 Therefore, stain is theTherefore, stain is the markermarker
or theor the reagentreagent used to generate the markerused to generate the marker

Why are stains taken into the tissues?Why are stains taken into the tissues?
 Dye tissue or reagent tissue affinitiesDye tissue or reagent tissue affinities
 Uptake of dyes or reagent is multistepUptake of dyes or reagent is multistep
Initial reaction – coulombic attractionInitial reaction – coulombic attraction
Later reaction – covalent bondingLater reaction – covalent bonding

 Involves 3 different interactionsInvolves 3 different interactions
1.1. Stain tissue interactionsStain tissue interactions
2.2. Solvent solvent interactionsSolvent solvent interactions
3.3. Stain stain interactionsStain stain interactions

Stain tissue interactionsStain tissue interactions
 Coulombic attractionsCoulombic attractions
 Van der waal’s forcesVan der waal’s forces
 Hydrogen bondingHydrogen bonding
 Covalent bondingCovalent bonding

Coulombic attractionsCoulombic attractions
 Also termed asAlso termed as
 saltsalt links orlinks or electrostaticelectrostatic bondsbonds
 Arise from electrostatic attraction of unlike ionsArise from electrostatic attraction of unlike ions
 Eg: basic dyes –phosphated DNA and RNAEg: basic dyes –phosphated DNA and RNA
Acid dyes – sulfated mucosubstancesAcid dyes – sulfated mucosubstances

 Dye ion binding depends onDye ion binding depends on
 Charge signs of dye and tissueCharge signs of dye and tissue
 MagnitudeMagnitude
 Amount of non dye electrolyte present inAmount of non dye electrolyte present in
dyebathdyebath
 Ability of tissue substrate to shrink or swellAbility of tissue substrate to shrink or swell

Such phenomena is important inSuch phenomena is important in
Best s carmine stainBest s carmine stain
When dyes go into the solutionWhen dyes go into the solution
TheyThey ionise and dissociateionise and dissociate
Acid dyes provide anions - chromogen
Positively charged ions - auxochromewww.indiandentalacademy.comwww.indiandentalacademy.com

 Reactive tissue groupings consist ofReactive tissue groupings consist of
 Bound moiety of one chargeBound moiety of one charge
 Mobile moiety of opp. ChargeMobile moiety of opp. Charge
 Tissue dissociates when immersed in dye bathTissue dissociates when immersed in dye bath
 Staining occurs….Staining occurs….
 When chromogen of one charge attracts the bound tissue moietyWhen chromogen of one charge attracts the bound tissue moiety
of other chargeof other charge

 Simple terms, reaction is as followsSimple terms, reaction is as follows
Tissue
NH2 Cl
Eosin
Chromogen Na
NNH2 chromogen Na Cl

Vanderwaal’s forcesVanderwaal’s forces
 Occur between reagent and tissue substratesOccur between reagent and tissue substrates
 Involves variousInvolves various intermolecularintermolecular attractionsattractions
 Dipole-dipoleDipole-dipole
 Dipole induced dipoleDipole induced dipole
 Dispersion forcesDispersion forces
 These forces are polar attractionsThese forces are polar attractions
 Effective over aEffective over a shortshort distancedistance
 Non symmetrical molecules posses stronger dipolesNon symmetrical molecules posses stronger dipoles
than symmetrical moleculesthan symmetrical moleculeswww.indiandentalacademy.comwww.indiandentalacademy.com

 Eg: staining ofEg: staining of elastic fibres by orceinselastic fibres by orceins
 Orcein is a large molecular weight dyeOrcein is a large molecular weight dye
 It has stronger dipolesIt has stronger dipoles
 Used in alcoholic solutionsUsed in alcoholic solutions
 Elastin is a hydrophobic proteinElastin is a hydrophobic protein
 Has many polarisable amino acidsHas many polarisable amino acids
 Hence the criteria for this force is metHence the criteria for this force is met

Hydrogen bondingHydrogen bonding
 Form of dye tissue attractionForm of dye tissue attraction
 Arises when hydrogen atom lies between two electronegative atomsArises when hydrogen atom lies between two electronegative atoms
 Spontaneous thermodynamic changes towardsSpontaneous thermodynamic changes towards disorganisationdisorganisation
leads to attraction between dye molecule and tissue groupsleads to attraction between dye molecule and tissue groups

 Eg: Best carmine s stain for glycogenEg: Best carmine s stain for glycogen
 Staining of amyloid and cellulose by variousStaining of amyloid and cellulose by various
bis azo dyesbis azo dyes
 In both the above examples , selective stainingIn both the above examples , selective staining
involves hydrogen bonding substitutes in the dyeinvolves hydrogen bonding substitutes in the dye
moleculesmolecules

Covalent bondingCovalent bonding
 Can occur between the stain and the tissueCan occur between the stain and the tissue
 In contrast to ionic bonding,In contrast to ionic bonding,
 Covalent bonding involves sharing of electronsCovalent bonding involves sharing of electrons
 Eg: in water, each of the 2 hydrogen atoms shares an electron withEg: in water, each of the 2 hydrogen atoms shares an electron with
oxygen, and the oxygen atom likewise shares two hydrogenoxygen, and the oxygen atom likewise shares two hydrogen
electronselectrons
 This bonding is of significance in mordant dying processThis bonding is of significance in mordant dying process

Solvent – Solvent InteractionSolvent – Solvent Interaction
 Hydrophobic bondingHydrophobic bonding
 It is the tendency of hydrophobic grouping to come togetherIt is the tendency of hydrophobic grouping to come together
 Water molecules held in clusters - hydrogen bondingWater molecules held in clusters - hydrogen bonding
 Transient clusters stabilised –hydrophobic groupsTransient clusters stabilised –hydrophobic groups
 Removing hydrophobic bonds – hydrophobic groups of substrateRemoving hydrophobic bonds – hydrophobic groups of substrate
and reagent becomesand reagent becomes markedmarked
 Eg: staining of fat by sudan dyesEg: staining of fat by sudan dyes

Stain- stain interactionsStain- stain interactions
 Dye molecule attracts each other – dye aggregatesDye molecule attracts each other – dye aggregates
 These are classic sites forThese are classic sites for metachromatic stainingmetachromatic staining
 Eg: toulidine blue – this effect is because dye aggregates haveEg: toulidine blue – this effect is because dye aggregates have
spectral properties different from monomeric dyesspectral properties different from monomeric dyes
 Eg: micro crystals of metallic silver in silver impregnationEg: micro crystals of metallic silver in silver impregnation

Why stains remain in tissue after removingWhy stains remain in tissue after removing
from stain bath?from stain bath?
 2 possibilities…2 possibilities…
 No affinity for processing fluids or mounting mediaNo affinity for processing fluids or mounting media
 Dissolves in these substances slowlyDissolves in these substances slowly
 Sections stained with basic dyes – should be dehydrated rapidlySections stained with basic dyes – should be dehydrated rapidly
 Sections stained with acidic or basic dyes are mounted in nonSections stained with acidic or basic dyes are mounted in non
aqueous media to prevent loss of dyeaqueous media to prevent loss of dye

Why are stains not taken up by every part ofWhy are stains not taken up by every part of
the tissue?the tissue?
FactorsFactors
1.1. Number and affinities of binding sitesNumber and affinities of binding sites
2.2. Rate of reagent uptakeRate of reagent uptake
3.3. Rate of reactionRate of reaction
4.4. Rate of reagent lossRate of reagent loss

Number and affinities of binding sitesNumber and affinities of binding sites
 Acid dyes – affinity for basic tissuesAcid dyes – affinity for basic tissues
 Basic dyes- affinity for acidic tissuesBasic dyes- affinity for acidic tissues
 This producesThis produces 2 tone2 tone staining pattern in which cytoplasm contraststaining pattern in which cytoplasm contrast
the nucleithe nuclei
 Affinities are influenced byAffinities are influenced by
 pHpH
 Concentration of inorganic saltConcentration of inorganic salt

Rate of reagent uptakeRate of reagent uptake
 Dyes diffusing at different rates exhibit staining rates ofDyes diffusing at different rates exhibit staining rates of
varying intensitiesvarying intensities
 Eg:Eg:
 red cellsstain slowlyred cellsstain slowly
 Collagen fibres stain rapidlyCollagen fibres stain rapidly
 Muscle fibres are intermediate in staining ratesMuscle fibres are intermediate in staining rates

Rate of reactionRate of reaction
 Selective staining depends on differential rates of reactionSelective staining depends on differential rates of reaction
 At low pH, hydrolysis of an organic phosphate is rapid in tissuesAt low pH, hydrolysis of an organic phosphate is rapid in tissues
containing acid phosphatasescontaining acid phosphatases
 Structures containing alkaline phosphatases, whose pH optima areStructures containing alkaline phosphatases, whose pH optima are
higher, the hydrolysis rates are slower.higher, the hydrolysis rates are slower.

Rate of reagent lossRate of reagent loss
 Factors affecting are:Factors affecting are:
1.1. Variation in section thicknessVariation in section thickness
2.2. TemperatureTemperature
3.3. Stirring of the reagent solutionStirring of the reagent solution
4.4. Presence of cavities in the tissuesPresence of cavities in the tissues
 Dyes are easily lost by permeable structuresDyes are easily lost by permeable structures
 Impermeable structures retain stain the longestImpermeable structures retain stain the longest

Effect of tissue modification on stainingEffect of tissue modification on staining
 Effects of fixationEffects of fixation
 May enhance or reduce the ionic strength of reactive groupsMay enhance or reduce the ionic strength of reactive groups
 Formalin and osmium tetroxide- induce basophiliaFormalin and osmium tetroxide- induce basophilia
 Acidic dichromate solutions – induce acidophiliaAcidic dichromate solutions – induce acidophilia

Effects of specimen geometry on staining?Effects of specimen geometry on staining?
 Varied 3D features of specimenVaried 3D features of specimen
1.1. Differences in few micro meter affects stainingDifferences in few micro meter affects staining
2.2. Dispersed cells stain differently compared to that cut from a blockDispersed cells stain differently compared to that cut from a block
3.3. Thin sections stain differently from thickThin sections stain differently from thick
4.4. Sections with irregular surfaces stain differently compared toSections with irregular surfaces stain differently compared to
smooth surfacesmooth surface

 Simple geometrical influence on stainingSimple geometrical influence on staining
1.1. Thin specimen stain faster than thickThin specimen stain faster than thick
2.2. Irregular surface stain faster than smoothIrregular surface stain faster than smooth
3.3. Dispersed specimen stain faster than smoothDispersed specimen stain faster than smooth

 Complex effects of specimen geometryComplex effects of specimen geometry
 Results from swelling of tissue componentsResults from swelling of tissue components
 Such swellings increase the rate of stainingSuch swellings increase the rate of staining
 This probably contributes toThis probably contributes to
 High selectivity of aqueous alcian blue for mucinsHigh selectivity of aqueous alcian blue for mucins
 High selectivity of strong acid picro trichrome stains for collagen fibresHigh selectivity of strong acid picro trichrome stains for collagen fibres

Effects of resin embedding on staining?Effects of resin embedding on staining?
 Resin as stain excludersResin as stain excluders
 Stain penetration is slowerStain penetration is slower
 Biological material is occluded – crooslinking increased –Biological material is occluded – crooslinking increased –
staining reducedstaining reduced
 Resin as stain bindersResin as stain binders
 Can give staining artefactsCan give staining artefacts
 Resin may reduce the amount of reagent reaching the targetResin may reduce the amount of reagent reaching the target
 Trap excess molecules – antiplasticising effectTrap excess molecules – antiplasticising effect

Thank you

Theories of staining (2) / dental implant courses by Indian dental academy

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