Staining G (1).pdf

HISTOPATHOLOGY AND CYTOLOGIC TECHNIQUES MLS MOD LECTURE/MARIA BENNETH PALEC, RMT, MSMT 3H 5G OUTLINE...

HISTOPATHOLOGY AND CYTOLOGIC TECHNIQUES MLS MOD LECTURE/MARIA BENNETH PALEC, RMT, MSMT 3H 5G OUTLINE I. Staining Principle: A. Principle Cationic (basic) dye stain acidic structures (nucleus, B. 3 Major Groups of Staining ribosomes) C. Terminologies Anionic (acid) dye stains basic structures (cytoplasm) II. Dye A. Dye Composition 3 Major Groups of Staining B. Classes of Dye 1. According to Origin 1. Histological Staining -microanatomicalsatiation of > cytoplasm nucleus. 2. According to Chemical configuration of the chromophore process whereby the tissue constituents are demonstrated 3. According to their electrical charge in sections by direct interaction with a dye or staining III. Staining Reactions - - solution reducing coloration of the active tissue components IV. Factors Affecting Staining aka Microanatomical Staining V. Types of Staining used to demonstrate the general relationship of tissue and cell w differentiation of nucleus and the cytoplasm - - LEARNING OUTCOMES example: Gomori trichrome blue stain At the end of this module, the student shall be able to: o used to stain and identify the muscle fibers, collagen 1. Explain the principles involved in staining of tissue section and nuclei 2. Define terms related to staining specific tissue substance. > 3. Appreciate the significance of each of the different special - 2. Histochemical Staining staining techniques sometimes called Histochemistry - - - various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a STAINING specific tissue substance process of applying dyes on the sections to see and study the example: pearl’s prussian blue reaction for Hb architectural pattern of the tissue and physical characteristics of example: periodic acid schiff staining for carbs the cells the last step of tissue processing is done to emphasize the features of the tissue 3. Immunohistochemical Staining staining the sections will allow the pathologists to study under combination of immunologic and histochemical the microscope the tissue samples techniques that allows phenotypic markers to be detected sections, as they are prepared, are colorless and demonstrated under the microscope Page | 1  MODULE 5D- STAINING OF TISSUE SECTIONS Terminologies example: phenol in carbo lithiation and carbolfuchsin o Does not participate in the staining reaction Trapping agent o Act by increasing the intensity, crispness and selectivity are chemicals which inhibit removal of dyes from the tissues of the dye rather than stop the removal of the dye completely, the usual o Not essential for the chemical union of the dye with the purpose is to slow it down so that tissues which are more tissue intensely stained still retain large amount of dye when the o Examples: ❖ Phenol - in carbolfuchsin and carbolthionine background has been decolorized ❖ Aniline - in gentian violet o this way, contrast has been enhanced ❖ Potassium hydroxide - in Loeffler’s methylene trapping agent – often confused with more dents, although blue the function is completely different Accelerators metallic impregnation - How to Differentiate Trapping Agent and Mordant? CNS Tissue Trapping agent Mordant Used in metallic impregnation techniques for the demonstration of CNS tissue components Usually non-metal Metals with a minimum valency of 2 Also acts as accentuators and they often consists of hypnotic Usually used with basic dyes Used with specific acid dyes drugs Example: barbital and chloral hydrate Is applied after the dye Usually applied before or with the dye Modifier > - exogenous Not required to complete No stain is possible if not One which when added to the dye will slightly alter the color of staining used a dye Inhibits removal of dye May be used to remove the Differ from the chromotrope in that they are exogenous by dye nature Examples: ❖ Methyl (CH₃) Most Common Example of Trapping Agent: ❖ Ethyl (C₂H₅) ✓ iodine in a gram stain, picric acid in gram Weigers method, ❖ Aryl (-C₆H₅) sodium chloride in salt gram technique Dye o Forms a complex with a dye ✓ possesses color because: o The complex is large and is “trapped” within the tissue or organism and is not readily released during 1. absorbs light in the visible spectrum of 400-700 nm differentiation 2. have at least one chromophore – color pairing group o Example: Gram’s iodine (complexes with crystal violet 3. have a conjugated system – a structure without tarnished during gram staining) double and single bonds 4. exhibits resonance of electrons – a stabilizing force in org Accentuator compounds does not participate in the staining reaction but merely accelerates or hastens the speed of the staining reaction by increasing the staining power and selectivity of the dye Page | 2  MODULE 5D- STAINING OF TISSUE SECTIONS NOTE: Examples of Natural Dyes 1. Hematoxylin Heartwood of the logwood tree  when any one of these features is lacking from the Derived from the heartwood of the logwood tree, molecular structure, the color is lost hematoxylin campechianum - -  addition to chromophores – most dyes also contain It is not a true dye until partially oxidized (ripened) - groups known as deoxochromes o By exposure to air - auxochrome – color helper o By chemical means Campech anum - , example: carboxylic acid, sulfonic acid, amino and hydroxyl groups Usually combined with alum or iron salt (acts as a - - mordant) to form a blue or black colored “lake”, respectively Leuco dye A colorless dye resulting from destruction of the chromotrope o M the most popular dye used as a nuclear - stain o develops the staining property after oxidation o a weak dye and to make it give a sharp stain, a DYE mordant is needed A colored aromatic salt which when in solution is capable of binding to another substance To avoid over-oxidation (and thus conversion into a Color index number (C.I.#) - a five-digit code used to indicate colorless leuco base), avoid exposure of hematoxylin the chemical configuration and color of the dye; dyes of (that has been ripened by chemical means) to air and purple potency violet > light - or decomposition identical structure made by different manufacturers and given - rust greenishor different names, will all be given the same color index number To test whether hematoxylin is still potent, add a drop of the stain to a beaker of water. A purple or violet color indicates M - Dye Composition - potency; any other color I(greenish > - or rust) Chromophore not permanent can therefore be easily removed , indicates decomposition - o The chemical group that imparts color to the dye 2. Carmine (carminic acid) Auxochrome Is a- -scarlet dye derived from the ground bodies of female o Gives a dye molecule its ionizing properties o It also provides “dyeing” properties and salt-forming cochineal bug, Coccus cacti - - - - properties - sativus - 3. Saffron Crocus CLASSES OF DYE Derived from the stigmas of a plant, Crocus sativus - - According to Origin Yellow-red to yellow in its dry orthochromatic state and is Natural yellow in solution Dyes extracted from plants, lichens or insects It is a “holy dye” used by the Buddhist monks to color their robes Their exact chemical composition is unknown, therefore they cannot be manufactured synthetically (very small in numbers) - - 4. Orcein Derived from lichen and requires oxidation to develop its blue-violet color Combined with a weak HCl, it is used for staining elastic fibers (as in van Gieson method) Page | 3  MODULE 5D- STAINING OF TISSUE SECTIONS tinstorm -bia 5. Alizarin Synthetic organic compounds containing the triphenyl Derived from the roots of the Madder plant Rubia methyl backbones (compounds that are intensely colored tinctorum and produced industrially) In Triarylmethane dyes, a central carbon is bonded to 3 6. Litmus aromatic rings, 1 is in the quininoid form Derived from a lichen - Very good example is the Malachite green – used as a Because of its poor histologic-staining property, it is used direct dye for wool and silk extensively as a pH indicator Basic Dyes Acidic Dyes - 7. Indigo Basic fuchsin Acid fuchsin oldest known dye Crystal violet Light green SF o blue dye indigo Methyl green Methyl blue both alizarin and indigo have very good dying properties Malachite green Aniline blue indigo remains as favored dye for denim (although Methyl violet synthetic indigo replaced the natural material) 2. Anthra-quininoid group Synthetic (Coal tar derivatives) Contain a quininoid ring which is the chromophore Coal tar derivative is a correct terminology for synthetic dyes These are derivatives of anthracene because all synthetic dyes are derived from coal tar and are Example: Alizarin red S - main ingredient for the benzene derivatives manufacture of madder late treatment known to painters Synthetic dyes usually have greater staining power and permit as rose madder and alizarin crimson a broader spectrum of color than natural dyes - Alizarin is used commercially as red textile dye - - cheaper to produce, brighter, more color fast and easy to Includes any groups of organic dyes having molecular structure based upon anthrax-quininoid apply to the fabric According to the chemical configuration of the chromophore 3. Xanthene group Quininoid chromophore group Important because of their brilliant use and shades A quininoid ring is a common chromophore between greenish yellow and purple Two atoms or groups having two valency bonds replace two Often fluorescent hydrogen atoms Most xanthene dyes are classified as basic dyes by their method of application The substituted groups must be in the ortho or para position Acid dyes can be produced by introduction of sulfonic acid groups Major groups of quininoid chromophore dyes Are derived from xanthene 1. Triarylamine group (triphenylmethane group) 3 major classes: This group of dyes has a quininoid ring chromophore o Eosin (acid dye) The structure of theses dyes contains 3 cyclic structures o Pyronin (basic dye) which have replaced the 4 hydrogen atoms of methane o Fluorescent dyes (CH₄) Page | 4  MODULE 5D- STAINING OF TISSUE SECTIONS 4. Hematein o Include: Sudan III, Sudan IV, Oil red O Is an amphoteric dye (carry both (+) and (-) charges o These dyes stain fats by a simple physical process Is the product of the oxidation of Hematoxylin (selective solubility) (C₁₆H₁₄O₆), a natural dye Trisazo and polyazo dyes o Contain 3 or more azo groups in their molecule 5. Quinone-imine group o Include: Triazo dye and Sirius light blue G Has a quininoid chromophore where one of the fundamental chromophoric groupings is C=N Nitro group Includes: The chromophore -NO₂ is found in the nitro group of the dyes o Indamines The chromophore is of such a strongly acid character that the o Indophenols dyes of this group are all acid dyes o Azines Picric acid (trinitrophenol) - the most common dye in this - - group Subdivisions o Thiazines Aliphatic compounds ▪ Have metachromatic properties These are linear hydrocarbons ▪ Include: basic dyes (thionine, azures, These are very rarely colored methylene blue and toluidine) o Oxazines Aromatic compounds o Azines These are hydrocarbons which include an “aromatic ring” ▪ Include: Basic dyes (neutral red and (arene) safranin O) Benzene-the simplest example Resonance of the electrons in these rings give the compound Azo chromophore dyes the property of color The chromophore of the azo group of dyes is the azo group (- Dyes are all aromatic compounds N=N-) Large class of synthetic organic dyes that contain nitrogen as Aniline dyes the azo group Aniline is a colorless coal-tar derivative Highly colored and prepared by diazothysine and aromatic amine and coupling with suitable aromatic compound It is the base from which many brilliant synthetic dyes are Can supply a complete rainbow of colors but the yellow-red made Aniline dyes offer a wide range in color and action dyes are more common than the blue-brown dyes The chemical composition of the dyes may be basic (cationic), acid (anionic) or neutral (amphoteric) Azo chromophore dye Subdivisions According to their electrical charge Monoazo dyes Reasonably easy to apply o Contain 1 azo coupling Have wide range of colors, and depending in dye selection can o Include: Orange G and Ponceau 2R have good color fastness property Typically used to dye natural proteins like wool and silk Disazo dyes o Contain 2 azo groups in the dye molecule Page | 5  MODULE 5D- STAINING OF TISSUE SECTIONS it is also use to dye synthetic polyamide like nylon and to a Examples of Basic Dyes small extent the acrylics and blends of these fibers (they are o Basic fuchsin o Crystal violet applied to these fibers from dye bonds in acidic or neutral o Carmine o Malachite green condition (carminic acid) o Methyl violet o Cresyl violet Acid Dyes It is a salt of a colored acid (sodium, potassium, calcium or ammonium salt) Amphoteric dyes The colored component (chromophore) is contained in the acid Have both positively and negatively chargeable groups on the component or the anion molecule The term “acid” does not refer to the hydrogen ion Depending on the charge actually present, this dye acts as either concentration (pH) of the dye solution positively or negatively charged ions In solution, acid dyes are anionic or negatively charged due to the determining factor in compounds of this nature including ionization of acidic auxochromes such as carboxyl, hydroxyl amino acids and proteins is the pH involved and sulphonate groups Such compound has a pH at which the overall charge on the Examples molecule is zero compound is considered to form an internal salt at this pH known o Acid fuchsin o Eosin B as zwitterion o Congo red o Eosin - - At pH levels below the isoelectric point, the positively charged o Aniline blue o Y Picric acid groups are favored and the compound is considered to be a cation t below the isoelectric point Basic dyes At pH levels above the isoelectric point, the negatively charged groups are favored and the compound is considered to be an Known as cationic dyes because the chromophore in basic anion - the above isoelectric point dimolecules contain a positive charge. act as bases and when made soluble in water they form a Has a point in its pH range where there is an equilibrium of colored cationic salt, which can react with the anionic sites in the surface of the substrate positive and negative charges. this is known as isoelectric point produce bright shades of high tinctorial values on textile (pI) materials A dye exhibiting amphoteric properties will act like a basic dye Not used on cotton, as the structures are neither planar, nor below the isoelectric point and act like an acid dye above the large enough for sufficient substantivity or affinity isoelectric point It is a salt of a colored base (chloride, acetates) which the dye Tissue proteins exhibit amphoteric properties stuff is the basic or positive radical Hematein is the only example The colored component (chromophore) is found in the base or cationic component Neutral dyes (Romanowsky’s dyes) Basic dye colors the nuclei, basophilic granules and bacteria Is a compound dye produced by combining an acid dye and a The acidic component of the tissue attracts the basic dyes basic dye in which both anion and cation are colored The term “basic” does not refer to the hydrogen ion Since the basic dye stains the nuclei whereas the acid dye concentration (pH) of the dye solution stains the cytoplasm a neutral dye would stain both the nuclei In solution, basic dyes are cationic or positively charged, due and cytoplasm to the ionization of the amino auxochrome Page | 6  MODULE 5D- STAINING OF TISSUE SECTIONS An acid dye and a basic dye are chemically combined and a Physical theories of staining precipitate is obtained Adsorption o The precipitate is known as a neutral dye The adsorbent tissue component enters into a loose It is insoluble in water but highly soluble in absolute alcohol combination with the material adsorbed (dye molecule) by Recommended for staining of blood films means of a weak intermolecular forces (i.e., hydrogen o Methanol is used as its solvent bonding or Van der Waals forces) o Ethanol causes hemolysis Examples: Hydrogen bonding – hydrogen being of single valency can o Giemsa’s stain only bond to another atom usually of those electronegative o Wright’s stain charge o May-Grunwald stain - occur readily in water - will occur between the dye and the water it is dissolved in STAINING REACTIONS - is of significant in alcoholic dye solution Absorption or direct staining Staining takes place without the help of other substances such Van der Waals forces – this intermolecular forces are polar as mordant or accentuator attractions The dye is absorbed by the tissue structure - weak and are effective over a short distance Occurs when a simple aqueous or alcoholic solution of a dye - attraction are between dipoles (molecules that are is applied to the tissue separated from positive and negative charges) Process of giving color to the sections by using aqueous or alcoholic dye solutions (e.g., methylene blue, eosin) Selective solubility This principle is employed in the selective staining of fats in oil- Indirect staining soluble dyes (lipochromes) The process whereby the reaction of the dye is intensified by Two main groups of oil-soluble dyes: adding another agent like the accentuator or mordant o Basic aryl amines - - Occurs when an intermediate substance (e.g. mordant) is ▪ With very low water solubility required in order for satisfactory staining to take place ▪ Examples: Sudan black B (most sensitive), The mordant binds with the dye to form a “lake” - Sudan Red VII B - serves as a link or bridge between the tissue and the o B-naphthols dye, to make the staining reaction possible ▪ Examples: Original diazo dyes (Sudan III, - very good example - potassium alum with hematoxylin Sudan IV (Scharlach B)) in Erich’s hematoxylin - iron in Weigert’s hematoxylin Porosity (permeability) o Mordant strengthens the bonding of the dye to the tissue structure The differential staining relies on the pore size of the protein o Mordant is employed in regressive staining because the precipitated meshwork and the dye molecule size differentiating agent breaks/threatens to break the Principle: electrical bond between the dye and the tissue During fixation, a protein precipitate and a meshwork is formed on the surface of the tissue (the pore size of the meshwork differ with the protein). The stain uptake of the tissue is a result of the permeability of the dye through the protein meshwork Page | 7  MODULE 5D- STAINING OF TISSUE SECTIONS produced on the surface of the tissue after treatment with Component of the fixative fixatives and other reagents. The dyes are applied in Since a fixative may exert an antagonistic or a synergistic chronological sequence. No differentiation is done in between effect during staining, a correct choice of fixative is therefore dye application. imperative o Zenker’s,10% buffered neutral formalin, Helly’s, Bouin’s Chemical theories of staining Susa, and Carnoy’s fluids permit a broad spectrum of staining methods Staining processes which involve the binding of dyes or dye o Zenker’s and Helly’s fixed tissues should be washed precursors to tissues by salt-linkages (ionic bonds), covalent thoroughly because mercury interferes with hematoxylin bonding, the formation of chelates, or the production of an staining insoluble pigment (i.e., Prussian blue) by a definite chemical o Formalin is not the best fixative for aniline dyes (trichome reaction, may be considered as “chemical theories of stain) and prolonged fixation in formalin inhibits eosin staining”. action - o chemical theories of staining – covalent bonding that - o Mercurial fixatives enhance nuclear staining and cellular occurs (involves sharing of electron) - - detail in general and is best used for trichome stain - o covalent bonding – is more significant than dyeing - - o Bouin’s is the best fixative in preparation for trichome Examples: - - - - stain - o Prussian blue method for demonstration of hemosiderin o Osmium tetroxide makes counterstaining almost - Bouin's ↳ Trichome impossible and its combination with chromate depresses Fe⁺⁺⁺ + potassium ferrocyanide → Ferric ferrocyanide (Prussian Stain. hematoxylin avidity (especially Ehrlich’s hematoxylin) blue color) o Buffered neutral formalin is the best fixative for the demonstration of iron pigments (hemosiderin) and for o PAS (periodic acid Schiff) reaction for polysaccharides elastic fibers which generally do not stain well after (detection of glycogen deposit) fixation with Susa, zenker’s or chromate fixatives Carbohydrate + PAS + Schiff’s reagent → magenta red color - Electric charge of the stain used Acid (anionic) dyes stain basic tissue structures; likewise, FACTORS AFFECTING STAINING basic (cationic) dyes stain acidic tissue structures The pH of the fixative The stain belongs to either acid or basic group not because of The pH of the fixative may change the pH of the tissue and its pH but because of the location of its chromophore group hence affect staining Generally, the pH of the fixative is maintained at a neutral pH Mordant so it won’t affect the stain uptake of the tissue Combines with the dye and the tissue to form a dye-mordant- Tissue fixation alters and reorganizes certain molecular tissue complex structures in tissue samples so that they have increased When mordant and dye are united, a substance known as a permeability and are more receptive in staining “lake” is formed Unfixed tissue elements have limited binding sites for dyes The “lake” is capable of combining with, and staining the tissue o the cells and other tissue elements often have an affinity The use of a mordant is very significant in regressive staining for stains or dyes with specific pH ranges where a decolorizer is used to remove excess stain. Since the o thus, the pH of the staining solution can have a direct decolorizer will act on the bond between the dye and the impact on the ability of the dye to bind with its intended tissue elements Page | 8  MODULE 5D- STAINING OF TISSUE SECTIONS tissue, the absence of a mordant will prevent the staining Presence of oxidizers and reducers (in the stain or in the tissue) reaction Hematoxylin is only a potential dye. It is ineffective unless strictly applicable to salts and hydroxides of divalent and oxidized to Hematein trivalent metals In metallic impregnation technique, the metallic salt is reduced example: alum or aluminum potassium sulfate into a metallic precipitate (silver precipitate) which settles on widely used mordant the surface of the tissue when acted upon by reducing chrome: potassium dichromate substances present on the tissue surface or in the staining brightens dye colors solution is commonly used in wools than any other fiber extremely toxic and therefore it should not be inhaled and TYPES OF STAINING gloves must be worn while working Regressive staining copper: copper sulfate used to bring out the greens in dyes The tissue is first overstained and then partially decolorized will also darken the dye colors When regressive staining is employed, a sharp degree of Examples: differentiation is obtained than with progressive staining o salts of metals (alum salts of aluminum, iron, chromium, copper, molybdenum, and vanadium) Steps in Regressive Staining o simple salts (sulfates and chlorides) of aluminum, iron, 1. Differentiation (decolorization) chromium, copper, molybdenum, and vanadium Is the removal or washing out of excess stain until the color is retained only by the tissue components that are Mordant Methods to be studied Pre-mordanting applied during fixation > -. It is the selective removal of stain o The mordant is applied during fixation - Accomplished with acid alcohol, ethyl alcohol, dye o Examples: picric acid (Bouin’s) mercury (Zenker’s for solvent/ diluention or the mordant Mallory’s Phosphomolybdic acid and Masson’s If the primary stain used is a basic dye, differentiation is Trichome stain) carried out by an acid solution, while alkaline medium is Post-mordanting > after formalin - used for differentiation after applying an acidic dye o The second fixative applied after formalin fixation acts - - Alcohol acts as a differentiator for both basic and acidic as a mordant - dye o Examples: picric acid and mercuric chloride for trichome staining Potassium dichromate (post- chromatization or post chromation) 2. “Blueing” Is the process of restoring the –OH (hydroxyl) ions lost Incorporating the mordant in the staining solution during the process of acid differentiation o Examples: Potassium alum, chromium alum, copper Acid differentiation removes the lake from the tissue alum (Celestine blue for nuclear staining) giving the tissue a red color This is done when alum hematoxylin is used during staining Includes a weak alkaline solution (Scott’s tap water substitute, lithium carbonate solution, weak ammonia water & alkaline tap water) Page | 9  MODULE 5D- STAINING OF TISSUE SECTIONS 3. Decolorization Two Methods of Vital staining Is the indiscriminate removal (partial or complete) of the 1. Intravital method stain from the tissue sections Accomplished by injecting the staining solutions into some parts of an animal body Progressive staining Demonstrates the Kupffer cells of the liver by intravenous The dye is applied in chronological order injection of Janus Green B Less favored than regressive staining due to the difficulty of Common dyes used: producing sufficiently intense progressive staining of cell o Lithium structures without staining other parts, thereby producing a o Carmine en diffused and obscure result a livingcu o India Ink a process whereby tissue elements are stained in definite sequence and the staining solution is applied for a specific 2. Supravital method examine Done by mixing the stain with living cells removed from period of time or until the desired intensity of coloring of the the animal body - different tissue elements is attained Demonstrates the reticulocytes using Methylene Blue N results to diffused color and obscured details due to difficulty Common dyes used: of producing sufficiently intense regressive staining of the cell o Neutral Red - best vital dye structure without staining the other parts once the dye is taken up by the tissue, it is not removed o Janus Green - for mitochondria. -.. o Trypan Blue Vital staining > - cell constituents living ▪ 1 gram dye is dissolved in 100 ml sterile cytoplasm distilled water; suspension must be use Is a selective staining of living cell constituents immediately, otherwise it is likely to become - - - Only the cytoplasm of the cell is stained - toxic to the cell The nucleus is resistant to vital stains - - o The demonstration of nuclear structures during vital Metachromatic staining staining suggests permeability of the membrane to the Entails the use of specific dyes which differentiates particular dye, signifying the death of the cell prior to the substances by giving them a color that is different from that of application of the vital stain the stain itself stains living cell by injecting the dye in any part of the animal Chromotrope: body A chemical found in certain tissues which has a capacity to done intravenously, intraperitoneally, subcutaneously polymerize the dye molecule resulting in the alteration of the o done to reduce specific coloration of certain cells color of the dye particularly those of the reticuloendothelial system Present in the Babes Ernst granules of C. diphtheria (stains red o intravenous in methylene blue), mast cell granules, cartilage and epithelial ▪ veins mucins (stains magenta red with toluidine blue) and amyloid o intraperitoneal (stains brilliant red with Congo red) ▪ through the peritoneum ▪ near stomach or abdominal cavity o subcutaneous All metachromatic dyes are basic dyes belonging to the ▪ through the skin Thiazine group and a Triarylamine (triphenylmethane) group Page | 10  MODULE 5D- STAINING OF TISSUE SECTIONS Thiazine group ▪ can also function as a staining agent Methylene blue Azure A Toluidine blue Azure B These metallic salts are then reduced to the opaque metallic Thionine Azure C form by: Triarylamine group 1. Argyrophyl reaction Love for silver Basic fuchsin Methylene violet “Love for silver” Crystal Violet Safranin A silver metallic impregnation technique which requires an Methyl violet Cresyl blue extraneous reducer to produce the black metallic silver at the site of the tissue component being demonstrated staining of the tissue elements in two colors from a solution of Examples: black metallic silver a single dye o reticulin fibers the two colors obtained are produced by the single chemical o nerve fibers compound attaching to the tissues in different fashions o Calcium salts dyes used to display metachromasia are basic dyes o Spirochetes NOTE: 2. Argentaffin reaction Metachromasia is lost if the section is dehydrated in alcohol “Affinity for silver” - Tissuecomponent after staining Hence why water differentiation is necessary for most Is a silver metallic impregnation technique where there is metachromatic staining techniques reduction to the silver salt by a tissue component - Substances capable of doing this are tyrosine derivatives and Metallic Impregnation phenolic compounds Involves the demonstration of tissue components resulting Examples: from the deposition of an opaque metallic residue on the o melanin actual structures o enterochromaffin cells (APUD cells) Metallic salts are used as the impregnating solution, the most o Argentaffin cells common being silver diamines and silver hexamines Tissues in argentaffin stain should not be fixed with an (methanamine) alcohol as they solubilize the argentaffin and decrease Salts of uranium, mercury, lead, gold and osmium can also be staining intensity used Melanin and certain other compounds A metallic impregnating agent is different from a stain in that: o they have sufficient reducing strength to reduce ionic silver to metallic silver in a basic solution o It is not absorbed by the tissue o Is held physically on the surface as a precipitate or as a reduction product in certain tissue components 3. Induced argentaffin reaction specific tissue elements are demonstrated not by stains but by The ability to reduce a silver salt can be induced into a tissue colorless solutions of metallic salts which are thereby reduced component which does not naturally have reducing properties by the tissue producing an opaque black deposit on the Example: surface of the tissue o reducing properties are introduced into fungi by utilized for silver staining of nervous system and also used to extensive oxidation in the Grocott’s technique demonstrate reticulin o example: gold chloride and silver nitrate ▪ most commonly used agent for impregnation Page | 11  MODULE 5D- STAINING OF TISSUE SECTIONS most fixatives could be used in routine H&E staining of paraffin Gold Toning embedded sections EXCEPT osmic acid solutions Type of Metallic Impregnation o because osmic acid soln inhibits hematoxylin The process of treating silver-impregnated section with gold chloride H&E staining of frozen sections for rapid diagnosis: Gold chloride turns silver into a gray precipitate o employs progressive staining Gold chloride eliminates the yellow background, gives greater o reagents are generally arranged in sequence using a transparency and improves differentiation series of coplin jars o takes only 5-10 minutes Solutions used for metallic impregnation: Special staining 1. Ammoniacal silver nitrate Demonstrates special features of the tissue such as bacteria, Used for the impregnation of reticulum, CNS tissues, and fungi, particular cell products, and microscopic intracellular argentaffin cells and intercellular structures Ammonium ion will combine with silver ion to produce a silver Examples: diamine or silver hexamine o Fuelgen stain for DNA Metachromatic staining 2. Aqueous silver nitrate Fluorescent staining Used for the impregnation of reticulum, CNS tissues, and The material is coupled with a dye which has a property of argentaffin cells fluorescing with ultraviolet light Fluorescence is the property of a substance to absorb light of 3. Sliver nitrate with methenamine (Gomori’s method) a shorter wavelength (e.g., UV light) and then emitting light of Used for demonstration of fungi, urates and basement a longer wavelength (visible light) membrane Fluorescence is observed under the fluorescent microscope Fluorescent dyes (fluorochromes) function as “tracers” in Routine staining immunopathologic studies One that stains the various tissue elements with little Examples: differentiation, except between nucleus and cytoplasm ❖ Acridine orange General relationships among cells, tissues and organs are ❖ Rhodamine B demonstrated ❖ Auramine O Examples: ❖ Sulforhodamine B o hematoxylin ❖ Fluorescein (MOST POPULAR) o eosin stains routine staining in paraffin embedded sections using H&E Trichome staining staining Used to differentiate 3 or more structures by giving each a employs regressive staining specific color o most common method utilized for microanatomical Applicable in differentiating collagen and elastic fibers from studies of tissues muscle fibers o consists of overstaining the nuclei, then removal of the excessive color of the tissue constituents by acid Utilizes the principle of porosity differentiation staining method in which two or more acid dyes of contrasting colors selectively collects different basic tissue components Page | 12  MODULE 5D- STAINING OF TISSUE SECTIONS used to demonstrate collagen, often in contrast to smooth APPENDICES muscle Bullet Legend: may also be used to emphasize fibrin in contrast to Info from the Video Lecture erythrocytes Info from the Module Packet use dyes in acid pH solvents o usually dilute aqueous acetic acid For more information, about Staining Tissue Processes: o acid pH – necessary to maximize the amount of the Gregorios,J.: Histopathologic Techniques, 2nd Ed., 2016 dye that will attach to the tissue amino group Dyes: https://www.britannica.com/technology/dye/General-features-of- dyes-and-dyeing. Counter staining Stains: https://www.youtube.com/watch?v=KSYyaxUgdns Is the application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated It is advantageous to use dilute solutions for counterstaining or to shorten the staining time Introduces color to specific cellular structure o to provide contrast to the colored enzyme substrate Aids in visualization and target localization, facilitating interpretation of morphology and cell structure within the tissue section Example: eosin counter stain in H&E stains Page | 13