OVERVIEW OF STAINING, .pdf

OVERVIEW OF THE THEORY OF STAINING BY B. A. OWUSU 8/12/24 OBRIGHTA 1  CHEMISTRY OF DYES INTRODUCTION Ø If unstained tissues are to be examined under light microscope, very little detail can be seen beyond the identification of cell boundaries and...

OVERVIEW OF THE THEORY OF STAINING BY B. A. OWUSU 8/12/24 OBRIGHTA 1  CHEMISTRY OF DYES INTRODUCTION Ø If unstained tissues are to be examined under light microscope, very little detail can be seen beyond the identification of cell boundaries and nucleus. Ø Dying or Staining can enable us to understand the physical characteristics and relations between tissue and their constituents 8/12/24 OBRIGHTA 2  STAINING AND IMPREGNATION Ø Cell and tissue components can be demonstrated not only by the use of stains but also by impregnation. These two methods differ in some respect: Ø Staining: cells and tissue components combine in molecular union with active colouring agent so that no particulate dye is seen and the tissues remain relatively transparent, unless very deeply staining. 8/12/24 OBRIGHTA 3  CONTINUATION Ø Impregnation: makes use of salts of heavy metals which are precipitated with considerable selectivity on certain cellular and tissue components. Ø The impregnation method has its greatest application in tissue from the nervous system but is also used for the demonstration of reticulin and, in the case of osmium tetroxide, for outlining fine intracellular structure. 8/12/24 OBRIGHTA 4  DIFFERENCES BETWEEN STAINING AND IMPREGNATION Ø Broadly impregnation differs from staining in that it consists of an opaque particulate precipitate. NB: The distinction is not absolute because in the final analysis, impregnation displays many of the characteristics of true staining. Ø Silver nitrate, which is the most commonly used agent for impregnation, can behave as a stain and outline the tissue elements in a non-particulate union. 8/12/24 OBRIGHTA 5  STAINING Ø Staining has been made possible by our understanding that different components of tissue have different affinities for different dyes. Ø Staining therefore depends on the physicochemical structure of dyes and tissues as well as the composition of cells and tissues. 8/12/24 OBRIGHTA 6  COLOURS IN STAINING Ø The wavelength of the visible spectrum of light for the human eye ranges between 400 and 750 nm Ø A proper mixture of all the wavelengths within this limits gives white light. Ø Therefore: In stained sections, the colour observed by the microscopist is the white light complement of wavelength minus the component absorbed by the section from the light passing through the tissue. 8/12/24 OBRIGHTA 7  EXAMPLES OF MICROSCOPIC OBSERVATIONS Ø If the wavelength 435-480 nm (blue-violet light) is absorbed by tissues, then the color seen by the microscopist is Yellow (picric acid). Ø Similarly, if yellow is absorbed then Blue is seen. Ø And if blue-green light (490-500nm) is absorbed, the colour seen is Red. 8/12/24 OBRIGHTA 8  DYES v Histochemical stains are dyes largely borrowed from textile industry. Obtained from two sources: a. Obtained from plants and animals (natural). b. Obtained from petrochemical industry (synthetic) 8/12/24 OBRIGHTA 9  CHEMISTRY OF SYNTHETIC DYES Ø Synthetic dyes are produced from colourless molecules usually with benzene ring as the central component Ø By introducing some chemical groups called chromophores to the molecules it will enable it to have a particular arrangement of atoms that will absorb light in the visible part of the spectrum Ø These are called chromogens Ø Benzene based molecule + chromophore = chromogen 8/12/24 OBRIGHTA 10  BENZENE RING HC CH HC CH HC CH 8/12/24 OBRIGHTA 11  EXAMPLES OF CHROMPHORES q-C=C q-NO2 qN=N- qC=O qC=S qC=N qN=O 8/12/24 OBRIGHTA 12  ANOTHER EXAMPLE OF CHROMOPHORES Quinonoid structure 8/12/24 OBRIGHTA 13  AUXOCHROMES Ø To turn chromogens into stains, it is necessary to introduce ionizing groups. This enables the dye molecule to have an affinity for the tissue. Ø It also confers the ability of electrolytic dissociation and salt formation to the molecule. This ionizing group is called an Auxochrome. Ø CHROMOGEN + AUXOCHROME = DYE/STAIN 8/12/24 OBRIGHTA 14  CONTINUATION Ø Auxochromes may be acidic (+ve charged) or basic (–ve charged). Ø Examples : Acidic groups: carboxyl -COOH hydroxyl -OH sulphydral –SO3H Basic groups: ammoniacal –NH3 8/12/24 OBRIGHTA 15  CLASSIFICATION OF DYES & MECHANISM OF STAINING 8/12/24 OBRIGHTA 16  CLASSIFICATION OF DYES Ø SOURCE Ø CHROMOPHORES Ø AUXOCHROMES (acids and bases or mordants) Ø MECHANISM OF STAINING Ø SUBSTRATES 8/12/24 OBRIGHTA 17  CLASSIFICATION BY SOURCE Ø NATURAL DYES: Plants and Animals Examples; hematoxylin, carmine, orcein Ø SYNTHETIC DYES: These synthetic dye groups may be classified in several groups. Example; Azo dyes, triphenylmethanes, acridine dyes 8/12/24 OBRIGHTA 18  CHROMOPHORES Ø Nitro dyes ------------picric acid, Martius yellow. Ø Azo dyes ---------------mono azo: orange g; diazo sudan IV Congo red. Ø Tetrazolium dyes-------Auramin O, MTT, XTT, NBT Ø Aryl methane dyes---diphenyl methane –crystal violet Ø Triaminotriphenomethane---- pyronin, rhodamine B Ø Xanthene dyes –-------------Amino- Eosin, Phloxine Ø Acridine dyes- ---------------acridine 8/12/24 OBRIGHTA 19  CONTINUATION Ø Quinolone/paraquinoid dyes------ hematoxylin Ø Thiazol dyes - ----------------------thioflavine T Ø Quinoneimine dyes- Ø Azin--------------------------------neutral red, safranin. Ø Oxazin---------------------------cresyl violet, Celestin Blue. Ø Thiazine -------------------------thionin, methylene blue Ø Anthraquinine- ------------------Alizarin red S, carmine Ø Phthalocyanins------------------ Alcian Blue, Luxol Fast Blue. 8/12/24 OBRIGHTA 20  AUXOCHROMES Classification of dyes by mechanism of staining Ø Acid dyes Ø Basic dyes Ø Neutral dyes Mordants (reactive) Mordants (disperse) Pigmenting dyes 8/12/24 OBRIGHTA 21  ACID DYES Ø In these it is the acidic radical that is the active or colouring agent, the basic part being inactive, e.g., acid fuchsin consist of the sodium salt of a sulfonate of rosaniline. 8/12/24 OBRIGHTA 22  BASIC DYES In these the active or colouring substance is a base and is combined with a colourless acidic radical, e.g., basic fuschin is the chloride salt of the base rosaline,. Generally, they stain acidic structures, e.g., cell nuclei. 8/12/24 OBRIGHTA 23  NEUTRAL DYES These consist of mixtures of basic and acidic dyes and therefore, usually consist of large molecular complexes which are very sparingly soluble in water and usually must be dissolved in alcohol. Examples are the Romanowsky dyes used in haematology. Both acidic and basic components retain their affinities for cell constituents of opposite reaction, and the whole compound dye stains neutrophilic structures. 8/12/24 OBRIGHTA 24  SUBSTRATES Ø Nucleic acids- phosphate backbone negatively charged is the reactive group. Ø Proteins – ionized amino grp NH3+, phenolic OH grp, SH, S-S- , Indole residue of tryptophan are all possible points for reactions. Ø Polysaccharides - Vicinal OH groups of CHO units can be used for dye attachments as in PAS stain Ø Lipids – free COOH, or C=C groups Ø Metals (inorganic salts) Fe, Ca Cu Mn, Al. ions can be detected using complexing agents e.g. Dithiooxamide with Cu, Cobalt, Ni ions. 8/12/24 OBRIGHTA 25  STAINING Biological staining is a union between a colored dye and a tissue substrate which resists simple washing. The union is weak so dyes may be recovered by appropriate methods. The mechanism depends principally on the structure of the dye molecule and the nature of the substrate with which it reacts. Also the solvent used can influence staining reactions. 8/12/24 OBRIGHTA 26  TYPES OF STAINING In staining the cells and tissues components combine in molecular union with the active colouring agent so that no particulate dye is seen and the tissue becomes transparent unless deeply stained. There are three types of staining; Ø Specific staining Ø Direct staining Ø In direct staining 8/12/24 OBRIGHTA 27  SPECIFIC STAINING Ø This is the basis of histochemistry, in which the identification of certain structures and chemical substances is accomplished by controlled, specific chemical reactions designed to give a final colour (staining) at the site or location of the structure or substance in the cells or tissues. Ø Such specific stains have little or no affinity for other tissue elements. Ø Examples include; demonstration of elastic fibers with Weigert’s elastic stain, glycogen with Best carmine, polysaccharides with the periodic acid-Schiff technique. 8/12/24 OBRIGHTA 28  DIRECT STAINING Ø This is the staining of tissue by means of simple solutions of dyes. 8/12/24 OBRIGHTA 29  INDIRECT STAINING The action of the dye is intensified by some other agent, that is a mordant. By itself, the dye may stain weakly if at all. The mordant may be incorporated in the staining solution or may be separated and, generally has the effect of enhancing the combination of the dyestuff with the tissues. 8/12/24 OBRIGHTA 30  MECHANISM OF STAINING Ø Ionic bond formation: a chemical bond in which electrons are transferred from one atom to another so that one bears a negative and the other a positive charge E.g. The negative charge of phosphate groups of nucleic acids are replaced by dye. Ø Covalent bonds: A chemical bond between two atoms or radicals formed by the sharing of electrons (single bond, double bond or triple bond). Ø The most permanent of staining processes, they are very strong and essentially irreversible under conditions encountered during staining. 8/12/24 OBRIGHTA 31  CONTINUATION Ø Electrostatic bonding: There are three types of electrostatic bond. These are: ion-ion interactions, ion-dipole interactions and dipole- dipole interactions Attraction between electron rich and electron deficient species on dyes and tissue components. The polar covalent bonds in the dyes enable: Ø Hydrogen bonds to form (O-H-----O-H) Van der Waal’s forces (three): I. Permanent dipole – dipole II. permanent dipole - induced dipole III. instantaneous induced dipole- induced dipole 8/12/24 OBRIGHTA 32  CONTINUATION Ø Hydrophobic bond (increase in entropy). The bond is formed because dye molecule and the substrate create a disorder in the system (entropy). Requires that binding takes place in aqueous solution and the dye and tissue both possess hydrophobic units. Ø *As the hydrophobic groups come together they break up the water clusters (from what is known as “flickering iceberg model”) 8/12/24 OBRIGHTA 33  OTHER STAINING MECHANISMS Ø Metachromasia; It is suggested that this is due to aggregates formed by hydrophobic bonding between dye molecules and tissue component (substrate). This results in alteration of spectra. Resulting in the staining of different tissue components different colors by same dye. Ø Fluorescent dyes; These are stains that emit light after electronic excitation. They however lack visible color and emit light as fluorescence when excited by UV light or a specific wavelength. The chemical structure of dye does not determine the mechanism of binding to substrate. 8/12/24 OBRIGHTA 34  CONTINUATION Ø Metal complexes: binding of dyes via metal complexes is an ancient technique known as mordanting. Ø Solvent dyes: these are dyes which are soluble in organic solvents. These have large non-polar (hydrophobic) conjugated systems and possess no richly polar solubilizing parts. (Weaker polar groups such as phenolic OH don’t seem to be involved in staining when present) Oil Red O, Sudan Black B Ø Pigment dyes: these are insoluble colored compounds which are generated within the substrate and remain trapped but not bonded. Alcian blue, Prussian blue, silver stains. Ø Immunostaining: specific antigen can be visualized by binding with an antibody labeled with chromogens. 8/12/24 OBRIGHTA 35  OTHER MECHANISMS. Ø Mixed stains: some common staining techniques use mixtures of two or more dyes. Example o Romanowsky – Giemsa stains. Consists of anion dyes eosin Y, and a cationic dye each as Azur A (or B) or methylene Blue. 8/12/24 OBRIGHTA 36  PROGRESSIVE AND REGRESSIVE STAINING Staining with haematoxylin (and many other dyes also) may be of two types: Ø Progressive staining; staining is continued until the desired intensity of colouring of the different tissue elements is attained. Ø Regressive staining; the tissues are over-stained and the excess dye is then removed selectively until the desired intensity is obtained. 8/12/24 OBRIGHTA 37  FACTORS AFFECTING DYE– TISSUE UNION Ø pH is critical in ionic bonding. Ø Molecular size: affects rate of diffusion. Chromophores with large hydrophobic groups tend to over stain and hence lack specificity. Ø Solvent effects: dimethyl sulphoxide swells tissues and disintegrates dyes , ethanol shrinks (dehydrates) and also disintegrates dyes 8/12/24 OBRIGHTA 38  CONTINUATION Ø Temperature: high temp increase rate of reaction and rate of diffusion Ø Surfactants: detergents counter surface tension, their presence as impurities in dye samples affect staining. Ø Time: The longer the time, the more intense the staining. Ø Light solubility of stains: excited molecules dissipate excess energy to return to ground state which can break up bonds that can result in loss of colour or photo fading due to breakdown of chromogenic structure. 8/12/24 OBRIGHTA 39  FACTORS THAT DETERMINE STAINING TIME Ø Type (natural or chemical ripened) and age of stain. Older stains require more time. Ø Type of tissue and how processed; paraffin or frozen, any pretreatment in acid, time in formalin or fixative Ø Progressive or regressive stain Ø Personal taste of the pathologist. 8/12/24 OBRIGHTA 40  FACTORS AFFECTING STAIN UPTAKE IN TISSUE v Selective staining uptake is fundamental to histochemistry, and even routine oversight methods such as hematoxylin and eosin (H&E), papanicolaou and Romanowsky-Giemsa stains distinguish nuclei from cytoplasm. 8/12/24 OBRIGHTA 41  CONTINUATION These include; Ø Numbers and affinities of binding sites; Stain-tissue affinities and numbers of binding sites present in tissues can vary independently. Sudan dyes can be used as an example. These have high affinity for fat but low affinity for the surrounding hydrated proteins. Ø Rates of reagent uptake: Progressive dyeing methods may be rate controlled, for instance mucin staining using alcian blue or colloidal iron. Selectivity requires short periods of dyeing during which only fast-staining mucins acquire colour. 8/12/24 OBRIGHTA 42  CONTINATION Ø Rates of reaction: Selective staining by reactive reagents, yielding coloured derivative, may depend on differential rates of reaction. Ø Rate of reagent loss: Differentiation or regressive staining involves selective losses of stain from tissues. Many dyeing methods exploit this phenomenon, e.g., staining of muscle striations with iron-hematoxylin. Ø Metachromasia and related phenomena: Even when neither affinity nor rate controls the staining pattern, selective coloration can still be obtained. 8/12/24 OBRIGHTA 43  EFFECTS OF DYE IMPURITIES ON STAINING. Ø An impure dye contains compound not named on the label or contains substantial amounts of other coloured substances additional to the named dye. Ø Most of the dyes used as stains are impure, which has provoked many experimental investigation. Ø Most impure batch contain very little dyes with most of the contents being inorganic salt. 8/12/24 OBRIGHTA 44  INFLUENCE OF IMPURITIES ON STAINS. There are two ways by which impurities influence stains and this include: 1. They alter staining intensity; typically staining is reduced, but very occasionally impurities result in a more intense colour. 2. They may change staining pattern; the nature and mechanisms of such effects depending on the type of impurity, the particular staining procedure, and the tissue substrate. 8/12/24 OBRIGHTA 45  SOLUTIONS TO DYE IMPURITIES ON STAINING. There is no simple way to identify impurities in stain. These practical tips would be of help. 1. Purchase dyes certified by the biological stains commission. 2. Check staining problem to know if it is due to impurities by retaining effective dye lots, if it gives satisfactory coloration. 8/12/24 OBRIGHTA 46  THE NOMENCLATURE OF DYES. Ø This includes names of individual dyes, and terms used to describe dye properties. Ø Nearly all dyes have trivial names that do not describe their structure. Example, Congo blue has its trivial name as tryptan blue. Ø Some nomenclature there is the surfeit of suffixes. Sometimes these are merely flourishes of a copywriter’s pen. Example is pyronines G and Y , which are synonyms. 8/12/24 OBRIGHTA 47  PROBLEM AVOIDANCE AND TROUBLESHOOTING. Ø Stains used must be compatible with the fixative and embedding medium. Example: water-miscible resin sections do not allow selective staining of elastic fibers with aldehyde fuchsin. Ø Use a routine, preferably a standardized, staining protocol. Ø Use control to detect problems proactively, not merely to investigate mistakes retrospectively. Note: Keep samples of effective batches of stain to use when you suspect inadequate stain purity. 8/12/24 OBRIGHTA 48  CUES FOR RECOGNISING ERRORS IN STAINING Ø Staining is not as expected in terms of colour, solubility or stability. Example: some alcian blue samples dissolve, but then precipitate from solution within an hour or less. Ø The expected structures stain, but only weakly. Examples: unexpectedly weak staining of calcium by alizarin red S results from extraction of tissue calcium ions into aqueous fixations. 8/12/24 OBRIGHTA 49  CONTINUATION Colour of staining is expected. Example: excessively red staining seen with Gomori’s trichrome may rise from insufficiently acidic staining solutions. Unexpected structures stain. Example: granular material stained by the Feulgen nuclear procedure may be carbonate deposits. Nature of the staining is unusual. Example: if differential staining of gram positive and negative organisms is poor, the preparation may be too thick. 8/12/24 OBRIGHTA 50  CONTINUATION Ø And there are always other problems! Examples: loss of sections from slides in the Grocott hexamine silver method for fungi, due to overheating; and black deposits on slides and sections in the Von Kossa procedure, due to contaminated glassware. NB: Once an error has been noticed, and a plausible cause identified, a solution can be sought. 8/12/24 OBRIGHTA 51  SOME IMPORTANT TERMINOLOGIES IN STAINING ACCENTUATORS Ø These are chemical substances which heighten the colour intensity, crispness, and selectivity of a stain. Ø They differ from mordants in that they do not bind or link the dye to the tissue. Ø Some appear to act as chemicophysical catalyst; others (e.g., aniline, and phenol) seem to work simply by reducing surface tension. 8/12/24 OBRIGHTA 52  DIFFERENTIATION Ø This process is the selective removal of excess dye. However in some staining techniques, e.g., with Romanowsky dyes, differentiation also implies the selective production of certain colours at specific pH values. Ø Differentiators for mordant dyes may be divided into three classes namely: a. Acids b. Oxidizers c. Mordants 8/12/24 OBRIGHTA 53  ACID DIFFERENTIATORS Ø These act by combining with the metal, thus breaking the latter’s union with the tissue or cell components. Ø The acid chosen should be one which forms a soluble salt with the metal so that the latter is dissolved out. Ø Examples are hydrochloric and acetic acids. 8/12/24 OBRIGHTA 54  OXIDIZING DIFFERENTIATORS Ø These act by oxidizing the dye to a colourless substance (leuco form). Components holding least dye will be bleached first. Ø Examples are potassium ferricyanide, potassium permanganate, chromic acid, picric acid, and potassium dichromate. Ø The last three are weak and slow differentiators. 8/12/24 OBRIGHTA 55  MORDANT DIFFERENTIATORS Ø When a section, which has been stained by a mordant dye, is placed in a solution of mordant, the latter is present in great excess and the dye gradually leaves the tissue to combine with the free mordant in solution. Ø Also mordants such as iron alum oxidize haematoxylin to a soluble colourless compound. 8/12/24 OBRIGHTA 56  CONTINUATION Ø Accordingly, the tissue components which contain the least dye will be decolorize first, and the structures containing most dye (nuclear chromatin) will still be deeply stained. Ø This heavy staining of the chromatin results from the results from the fact that the dye-mordant complexes are basic and unite preferentially with the acidic nuclear structures, especially if salts of aluminium are used as mordant. 8/12/24 OBRIGHTA 57  DIFFERENTIATORS AS DECOLORIZERS Ø Sections which has been stained by a mordant dye is allowed to remain long enough in a differentiator, e.g., 1 to 2% acid alcohol, all the dye will be removed. Ø Indeed, this is actually done as a preliminary step in the restaining of a faded slide. NB: accordingly, in routine staining, care must be taken that sections are not left too long in differentiators. 8/12/24 OBRIGHTA 58  BLUEING Alum (potassium aluminium sulphate) in watery solution tends to dissociate: the aluminium combines with the –OH of the water to form insoluble aluminium hydroxide, Al(OH)3; the free hydrogen from the water tends to form sulfuric or other acid by uniting with the sulphate from the alum. 8/12/24 OBRIGHTA 59  CONTINUATION Ø However, if excess of acid (sulfuric or other acid) is present, the aluminium hydroxide cannot form. Under such circumstances, in an alum hematoxylin dye the insoluble dye lake cannot form because of lack of hydroxyl ions. Ø In blueing sections which have been stained by an alum hematoxylin, the alkaline solution used for blueing neutralises the free acid and makes the hydroxyl group available so that the insoluble blue aluminium –hematein –tissue lake is formed. 8/12/24 OBRIGHTA 60  CONTINUATION Ø Accordingly, for blueing of alum-haematoxylin, stained sections warm (40-50℃.) tap water is commonly used since is generally sufficiently alkaline. Ø However, in many areas the tap water is acid and not suitable. 8/12/24 OBRIGHTA 61  THANK YOU, ANY QUESTIONS?? 8/12/24 OBRIGHTA 62