1. Hematoxylin is a natural dye extracted from logwood that is used as a nuclear stain. It requires oxidation to hematein and use of a mordant like aluminum or iron to effectively stain nuclei.
2. Alum hematoxylin is commonly used, with Ehrlich's, Harris', and Mayer's being examples. It stains nuclei blue-black but is sensitive to acid. Iron hematoxylin is more resistant.
3. Oxidation can be done naturally over months or chemically instantly using sodium iodate or mercuric oxide. This affects the staining properties and lifespan.
This document discusses hematoxylin and eosin stains, which are commonly used histological stains. It describes the key components - hematoxylin stains cell nuclei blue or black, while eosin stains cytoplasm and connective tissues in shades of pink, orange and red. Hematoxylin is extracted from logwood and oxidized to hematin, its active form, which requires a mordant like aluminum or iron salts to bind to tissue. Different hematoxylin types and methods are classified. Alum hematoxylin is most routinely used and its progressive staining method is described along with the processes of differentiation, bluing, and potential deterioration over time. Other mordant types and their uses are also mentioned
Hematoxylin and eosin staining is a common histological technique that uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The document describes the full H&E staining procedure, including dewaxing, hydration, staining, differentiation, dehydration, clearing and mounting of tissue sections. It also discusses the principles and properties of hematoxylin, including how it is extracted from logwood and requires oxidation or "ripening" to become an effective nuclear stain. Commonly used hematoxylin formulations including Harris's, Mayer's, and Ehrlich's are compared.
This document discusses hematoxylin and eosin stains. It provides details on:
1) Hematoxylin is extracted from logwood and oxidized to hematin, which is responsible for staining properties. It requires a mordant like aluminum or iron salts to bind to tissues.
2) Alum hematoxylin is commonly used, with potassium or ammonium alum as the mordant. Sections can be overstained and differentiated, or stained for a predetermined time.
3) After differentiation, sections are "blued" in a weak alkaline solution to convert the hematin stain from red to blue-black in the cell nuclei.
The document summarizes hematoxylin and eosin staining. Hematoxylin is extracted from logwood and stains cell nuclei blue-black by binding to DNA and RNA. It requires a mordant like aluminum or iron to bind to tissues. The staining process involves hematoxylin ripening, which can be done naturally or chemically using oxidizing agents. Different hematoxylin formulations use different mordants and oxidation methods. Hematoxylin is then differentiated using an acid to remove excess stain from cytoplasm before bluing and mounting. Common hematoxylin stains discussed include Ehrlich's, Mayer's, Harris', and Gill's.
The tissue section is colourless because the fixed protein has the same refractive index as that of glass. We use dyes that have specific affinity with the different tissue proteins and colour them differently.
Colour is seen by the eye as a result of the effect of certain electromagnetic waves on the rods and cones of the retina. These waves, which have a varying length, will determine the colour that is seen.
White light being composed of all the colours of the visible spectrum varies in wavelength from 4,000 Â to 8,000 Â.
If light of a specific wavelength is absorbed from white light the resultant light will then be coloured, the colour being dependent upon the particular wavelength that has been removed.
The document discusses the hematoxylin and eosin stain, which is the most widely used histological stain. It stains cell nuclei blue or black using hematoxylin, and stains cell cytoplasm and connective tissue fibers pink using eosin. The purpose of staining is to identify tissue structures and the presence or absence of disease. Common stains discussed include hematoxylin and eosin, Gram's method, Ziehl-Neelson's method, and Papanicolaou stain. The document also provides details on the chemistry and procedures for hematoxylin and eosin staining.
Histological staining methods aim to differentiate tissues and cells by altering contrast or color. There are several staining techniques including vital staining of living cells, staining by selective solubility of dyes in lipids, chemical reactions between dyes and tissues, metallic impregnation, and staining with synthetic or natural dyes. Dyes contain chromophores for color and auxochromes that determine staining action. Staining can be progressive, with sequential coloring of elements, or regressive with initial overstaining followed by differentiation. Factors like dye concentration, temperature, pH, fixation influence dye binding to tissues.
This document discusses hematoxylin and eosin stains, which are commonly used histological stains. It describes the key components - hematoxylin stains cell nuclei blue or black, while eosin stains cytoplasm and connective tissues in shades of pink, orange and red. Hematoxylin is extracted from logwood and oxidized to hematin, its active form, which requires a mordant like aluminum or iron salts to bind to tissue. Different hematoxylin types and methods are classified. Alum hematoxylin is most routinely used and its progressive staining method is described along with the processes of differentiation, bluing, and potential deterioration over time. Other mordant types and their uses are also mentioned
Hematoxylin and eosin staining is a common histological technique that uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The document describes the full H&E staining procedure, including dewaxing, hydration, staining, differentiation, dehydration, clearing and mounting of tissue sections. It also discusses the principles and properties of hematoxylin, including how it is extracted from logwood and requires oxidation or "ripening" to become an effective nuclear stain. Commonly used hematoxylin formulations including Harris's, Mayer's, and Ehrlich's are compared.
This document discusses hematoxylin and eosin stains. It provides details on:
1) Hematoxylin is extracted from logwood and oxidized to hematin, which is responsible for staining properties. It requires a mordant like aluminum or iron salts to bind to tissues.
2) Alum hematoxylin is commonly used, with potassium or ammonium alum as the mordant. Sections can be overstained and differentiated, or stained for a predetermined time.
3) After differentiation, sections are "blued" in a weak alkaline solution to convert the hematin stain from red to blue-black in the cell nuclei.
The document summarizes hematoxylin and eosin staining. Hematoxylin is extracted from logwood and stains cell nuclei blue-black by binding to DNA and RNA. It requires a mordant like aluminum or iron to bind to tissues. The staining process involves hematoxylin ripening, which can be done naturally or chemically using oxidizing agents. Different hematoxylin formulations use different mordants and oxidation methods. Hematoxylin is then differentiated using an acid to remove excess stain from cytoplasm before bluing and mounting. Common hematoxylin stains discussed include Ehrlich's, Mayer's, Harris', and Gill's.
The tissue section is colourless because the fixed protein has the same refractive index as that of glass. We use dyes that have specific affinity with the different tissue proteins and colour them differently.
Colour is seen by the eye as a result of the effect of certain electromagnetic waves on the rods and cones of the retina. These waves, which have a varying length, will determine the colour that is seen.
White light being composed of all the colours of the visible spectrum varies in wavelength from 4,000 Â to 8,000 Â.
If light of a specific wavelength is absorbed from white light the resultant light will then be coloured, the colour being dependent upon the particular wavelength that has been removed.
The document discusses the hematoxylin and eosin stain, which is the most widely used histological stain. It stains cell nuclei blue or black using hematoxylin, and stains cell cytoplasm and connective tissue fibers pink using eosin. The purpose of staining is to identify tissue structures and the presence or absence of disease. Common stains discussed include hematoxylin and eosin, Gram's method, Ziehl-Neelson's method, and Papanicolaou stain. The document also provides details on the chemistry and procedures for hematoxylin and eosin staining.
Histological staining methods aim to differentiate tissues and cells by altering contrast or color. There are several staining techniques including vital staining of living cells, staining by selective solubility of dyes in lipids, chemical reactions between dyes and tissues, metallic impregnation, and staining with synthetic or natural dyes. Dyes contain chromophores for color and auxochromes that determine staining action. Staining can be progressive, with sequential coloring of elements, or regressive with initial overstaining followed by differentiation. Factors like dye concentration, temperature, pH, fixation influence dye binding to tissues.
This document provides an overview of tissue fixation techniques. It defines fixation as a process that preserves tissues in a state close to how they appeared when living. This is achieved by preventing autolysis and maintaining cellular morphology. The document discusses various types of fixatives including aldehydes, alcohols, and oxidizing agents. It also covers the aims of fixation, how different fixatives work, commonly used fixatives for different tissue and cellular components, and potential artifacts. Fixation is essential for histological examination and aims to maintain tissues for further analysis.
The Hematoxylin and Eosin stain is the most widely used histological stain. It clearly demonstrates many tissue structures using a simple method. Hematoxylin stains cell nuclei blue-black, while Eosin stains cytoplasm and connective tissues in shades of pink, orange, and red. This allows for the identification of tissues and the detection of disease processes under the microscope. The stain involves coloring the sample with hematoxylin, differentiating with an acid, and counterstaining with eosin.
This document discusses histopathology staining techniques. It defines histopathology as staining tissue samples to examine cellular and intracellular structures microscopically. The two main categories of stains are natural dyes derived from natural resources, and artificial dyes produced through chemical reactions. Hematoxylin is a commonly used natural dye that stains nuclei blue, while eosin is an artificial dye that counterstains cytoplasm pink. The document outlines hematoxylin and eosin staining as well as other histological stains and their characteristics.
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This document provides an overview of various special staining techniques used in histology, including:
1. Trichrome stains like Masson's and van Gieson's which stain nuclei, cytoplasm, muscle fibers, and collagen different colors.
2. Metal impregnation techniques like Gordon and Sweets' silver stain to demonstrate reticular fibers.
3. Carbohydrate stains like Periodic acid-Schiff to identify glycogen, mucins, and basement membranes.
4. Hematoxylin and eosin, Gram stain, and Ziehl-Neelsen to identify cells, bacteria, and acid-fast bacteria respectively.
5. Additional specialized stains for
- Hematoxylin and eosin (H&E) staining is the most widely used staining technique in histopathology. Hematoxylin stains cell nuclei blue-black, while eosin stains cytoplasm and other tissues shades of pink-red.
- Hematoxylin is extracted from the logwood tree and must be oxidized to hematin to function as a stain. It is used with a mordant like aluminum or iron salts to bind to cell nuclei. Alum hematoxylins are the most common and produce red nuclei that are "blued" after staining.
- Different hematoxylin solutions exist based on the mordant used, including alum, iron,
Hematoxylin and eosin (H&E) staining is the most common histological staining method. Hematoxylin stains cell nuclei blue by combining with oxidized hematin and a mordant like alum. Eosin stains cytoplasm and extracellular substances pink. For H&E staining, tissue sections are stained in hematoxylin, rinsed in acid alcohol to differentiate nuclei, rinsed in water to turn nuclei blue, and then stained in eosin to color non-nuclear structures pink, allowing easy visualization of cell morphology. H&E staining provides essential structural information and is useful for pathology examinations.
This document provides an overview of hematoxylin and eosin staining techniques. It discusses the structures of dyes, classifications of dyes, staining mechanisms, hematoxylin, eosin, and the hematoxylin and eosin staining procedure. The key steps of the H&E staining procedure are deparaffinization, hydration, hematoxylin staining, differentiation, blueing, and eosin counterstaining to color nuclei blue and cytoplasm pink, respectively. The document also covers dye origins, properties, affinities, and terms used in biological staining.
Pigments and minerals in tissue can be classified as artificial, exogenous, or endogenous. Artificial pigments are fixation artifacts from chemicals like mercury or chrome. Exogenous pigments originate outside the body from sources like carbon, silica, or tattoo pigments. Endogenous pigments are produced within tissues and include hematogenous pigments derived from hemoglobin breakdown like hemosiderin, and non-hematogenous pigments like melanin, lipofuscin, and porphyrin. Special stains are used to identify and characterize various pigments based on their properties.
This document discusses the process of decalcification, which is the removal of calcium salts from bones and calcified tissues. There are four main methods: 1) using simple dilute mineral acids like nitric acid or formic acid; 2) ion exchange resins with acid fluids; 3) electrolytic decalcification using electrodes; and 4) chelating agents like EDTA that bind calcium ions. The document provides details on procedures and advantages/disadvantages of each method.
This document discusses cytopreparatory techniques, including fixation of cytological samples, staining methods, and interpretation. It focuses on fixation, explaining that fixation preserves cells in a lifelike state after death by preventing autolysis and putrefaction. The key properties of a good fixative are outlined, and various fixatives are classified and examples are provided, including alcohols, formalin, and mercuric chloride, which are commonly used for cytological preparations.
THEORIES OF STAINING Biological Staining
Structural Components (Nature) Of Stains
Staining Mechanism
Metachromasia
Types Of Staining
Staining of Paraffin Section
A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.
The term ‘dye’ is used to refer to a colouring agent that is used for general purposes, whereas the term ‘stain’ is used to refer to that dye which is used for biological purposes.
The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2).
The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin, etc.FACTORS INFLUENCING METACHROMASIA
Frozen sections of tissue are prepared using a cryostat to quickly obtain thin sections for histological examination and diagnosis. A cryostat maintains tissue at freezing temperatures to allow sectioning without ice crystal formation. Tissue is mounted on a chuck and placed in the cryostat, then sectioned and mounted on slides for staining. Frozen sections allow rapid diagnosis but have poorer morphology and staining than fixed tissue sections.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
This document discusses various staining techniques used to differentiate tissues and cellular structures under a microscope. It describes the objectives of staining as revealing internal and external structures and producing specific chemical and physical reactions. The 3 major groups of staining are histological, histochemical, and immunohistochemical staining. Histological staining uses dyes to demonstrate tissue and cell relationships. Histochemical staining localizes specific tissue substances through chemical reactions. Immunohistochemical staining uses antibodies to detect phenotypic markers. The document outlines different types of stains, staining methods like direct, indirect and regressive staining, as well as procedures for staining frozen sections, paraffin sections, and broken slides.
This document discusses histopathology and the process of tissue fixation. It defines histopathology as the study of diseased tissues to examine changes in structure from disease. The key steps in tissue fixation are described, including the objectives to preserve tissue structure and prevent decomposition. Various types of fixatives are classified and their mechanisms and properties explained, with examples like formalin, glutaraldehyde, alcohol, picric acid and osmium tetraoxide. Compound fixatives are also mentioned.
Lecture (4) commonly used fixatives in the laboratoryHafsa Hussein
This document discusses various chemical fixatives used to preserve tissue samples for microscopic examination, including their components, tissues they are suitable for, advantages, and disadvantages. Bouin's fixative contains picric acid and formaldehyde and is excellent for glycogen but damages nucleic acids. Mercury-containing fixatives like Zenker's fluid and Helly's fluid act rapidly but are toxic. Fixation artifacts can occur if issues arise during the fixation process and obscure tissue details. The document provides methods for removing various artifacts that may occur.
This document provides information on techniques for collecting, preparing, preserving, and displaying specimens in a pathology museum. It discusses receiving specimens from hospitals and laboratories, preparing them by washing and fixing in formalin-based solutions, restoring color with alcohol, and long-term preservation by mounting in glycerin-based solutions. Special techniques are described for various organs and structures like lungs, brains, and calculi. The goal is to maintain specimens in a lifelike state for teaching and display over many years.
GENERAL PRINCIPLES OF STAINING AND H & E STAIN.pptxDr.Shubham Patel
This document provides an overview of general principles of staining and the hematoxylin and eosin (H&E) stain. It discusses why staining is necessary, common terminology used, theories of staining, types of stains and dyes, the roles of mordants and accentuators, and metachromasia. It then focuses on H&E stain, including the history and development of hematoxylin, the staining process, and different types of hematoxylin classifications based on the oxidation procedure and mordant used.
The document discusses the theory of staining in histology. It explains that staining allows tissues to be distinguished by providing different colors. Staining works through interactions between dyes and tissue components based on factors like charge, size, and chemical bonds. Differential staining is achieved through differences in binding capacity, rate, and strength between dyes and tissues. The document outlines various dye properties, classification systems, and staining methods. It also addresses factors influencing staining quality and provides examples of common problems and their solutions.
This document provides an overview of tissue fixation techniques. It defines fixation as a process that preserves tissues in a state close to how they appeared when living. This is achieved by preventing autolysis and maintaining cellular morphology. The document discusses various types of fixatives including aldehydes, alcohols, and oxidizing agents. It also covers the aims of fixation, how different fixatives work, commonly used fixatives for different tissue and cellular components, and potential artifacts. Fixation is essential for histological examination and aims to maintain tissues for further analysis.
The Hematoxylin and Eosin stain is the most widely used histological stain. It clearly demonstrates many tissue structures using a simple method. Hematoxylin stains cell nuclei blue-black, while Eosin stains cytoplasm and connective tissues in shades of pink, orange, and red. This allows for the identification of tissues and the detection of disease processes under the microscope. The stain involves coloring the sample with hematoxylin, differentiating with an acid, and counterstaining with eosin.
This document discusses histopathology staining techniques. It defines histopathology as staining tissue samples to examine cellular and intracellular structures microscopically. The two main categories of stains are natural dyes derived from natural resources, and artificial dyes produced through chemical reactions. Hematoxylin is a commonly used natural dye that stains nuclei blue, while eosin is an artificial dye that counterstains cytoplasm pink. The document outlines hematoxylin and eosin staining as well as other histological stains and their characteristics.
The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.for more details please visit
www.indiandentalacademy.com
This document provides an overview of various special staining techniques used in histology, including:
1. Trichrome stains like Masson's and van Gieson's which stain nuclei, cytoplasm, muscle fibers, and collagen different colors.
2. Metal impregnation techniques like Gordon and Sweets' silver stain to demonstrate reticular fibers.
3. Carbohydrate stains like Periodic acid-Schiff to identify glycogen, mucins, and basement membranes.
4. Hematoxylin and eosin, Gram stain, and Ziehl-Neelsen to identify cells, bacteria, and acid-fast bacteria respectively.
5. Additional specialized stains for
- Hematoxylin and eosin (H&E) staining is the most widely used staining technique in histopathology. Hematoxylin stains cell nuclei blue-black, while eosin stains cytoplasm and other tissues shades of pink-red.
- Hematoxylin is extracted from the logwood tree and must be oxidized to hematin to function as a stain. It is used with a mordant like aluminum or iron salts to bind to cell nuclei. Alum hematoxylins are the most common and produce red nuclei that are "blued" after staining.
- Different hematoxylin solutions exist based on the mordant used, including alum, iron,
Hematoxylin and eosin (H&E) staining is the most common histological staining method. Hematoxylin stains cell nuclei blue by combining with oxidized hematin and a mordant like alum. Eosin stains cytoplasm and extracellular substances pink. For H&E staining, tissue sections are stained in hematoxylin, rinsed in acid alcohol to differentiate nuclei, rinsed in water to turn nuclei blue, and then stained in eosin to color non-nuclear structures pink, allowing easy visualization of cell morphology. H&E staining provides essential structural information and is useful for pathology examinations.
This document provides an overview of hematoxylin and eosin staining techniques. It discusses the structures of dyes, classifications of dyes, staining mechanisms, hematoxylin, eosin, and the hematoxylin and eosin staining procedure. The key steps of the H&E staining procedure are deparaffinization, hydration, hematoxylin staining, differentiation, blueing, and eosin counterstaining to color nuclei blue and cytoplasm pink, respectively. The document also covers dye origins, properties, affinities, and terms used in biological staining.
Pigments and minerals in tissue can be classified as artificial, exogenous, or endogenous. Artificial pigments are fixation artifacts from chemicals like mercury or chrome. Exogenous pigments originate outside the body from sources like carbon, silica, or tattoo pigments. Endogenous pigments are produced within tissues and include hematogenous pigments derived from hemoglobin breakdown like hemosiderin, and non-hematogenous pigments like melanin, lipofuscin, and porphyrin. Special stains are used to identify and characterize various pigments based on their properties.
This document discusses the process of decalcification, which is the removal of calcium salts from bones and calcified tissues. There are four main methods: 1) using simple dilute mineral acids like nitric acid or formic acid; 2) ion exchange resins with acid fluids; 3) electrolytic decalcification using electrodes; and 4) chelating agents like EDTA that bind calcium ions. The document provides details on procedures and advantages/disadvantages of each method.
This document discusses cytopreparatory techniques, including fixation of cytological samples, staining methods, and interpretation. It focuses on fixation, explaining that fixation preserves cells in a lifelike state after death by preventing autolysis and putrefaction. The key properties of a good fixative are outlined, and various fixatives are classified and examples are provided, including alcohols, formalin, and mercuric chloride, which are commonly used for cytological preparations.
THEORIES OF STAINING Biological Staining
Structural Components (Nature) Of Stains
Staining Mechanism
Metachromasia
Types Of Staining
Staining of Paraffin Section
A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.
The term ‘dye’ is used to refer to a colouring agent that is used for general purposes, whereas the term ‘stain’ is used to refer to that dye which is used for biological purposes.
The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2).
The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin, etc.FACTORS INFLUENCING METACHROMASIA
Frozen sections of tissue are prepared using a cryostat to quickly obtain thin sections for histological examination and diagnosis. A cryostat maintains tissue at freezing temperatures to allow sectioning without ice crystal formation. Tissue is mounted on a chuck and placed in the cryostat, then sectioned and mounted on slides for staining. Frozen sections allow rapid diagnosis but have poorer morphology and staining than fixed tissue sections.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
This document discusses various staining techniques used to differentiate tissues and cellular structures under a microscope. It describes the objectives of staining as revealing internal and external structures and producing specific chemical and physical reactions. The 3 major groups of staining are histological, histochemical, and immunohistochemical staining. Histological staining uses dyes to demonstrate tissue and cell relationships. Histochemical staining localizes specific tissue substances through chemical reactions. Immunohistochemical staining uses antibodies to detect phenotypic markers. The document outlines different types of stains, staining methods like direct, indirect and regressive staining, as well as procedures for staining frozen sections, paraffin sections, and broken slides.
This document discusses histopathology and the process of tissue fixation. It defines histopathology as the study of diseased tissues to examine changes in structure from disease. The key steps in tissue fixation are described, including the objectives to preserve tissue structure and prevent decomposition. Various types of fixatives are classified and their mechanisms and properties explained, with examples like formalin, glutaraldehyde, alcohol, picric acid and osmium tetraoxide. Compound fixatives are also mentioned.
Lecture (4) commonly used fixatives in the laboratoryHafsa Hussein
This document discusses various chemical fixatives used to preserve tissue samples for microscopic examination, including their components, tissues they are suitable for, advantages, and disadvantages. Bouin's fixative contains picric acid and formaldehyde and is excellent for glycogen but damages nucleic acids. Mercury-containing fixatives like Zenker's fluid and Helly's fluid act rapidly but are toxic. Fixation artifacts can occur if issues arise during the fixation process and obscure tissue details. The document provides methods for removing various artifacts that may occur.
This document provides information on techniques for collecting, preparing, preserving, and displaying specimens in a pathology museum. It discusses receiving specimens from hospitals and laboratories, preparing them by washing and fixing in formalin-based solutions, restoring color with alcohol, and long-term preservation by mounting in glycerin-based solutions. Special techniques are described for various organs and structures like lungs, brains, and calculi. The goal is to maintain specimens in a lifelike state for teaching and display over many years.
GENERAL PRINCIPLES OF STAINING AND H & E STAIN.pptxDr.Shubham Patel
This document provides an overview of general principles of staining and the hematoxylin and eosin (H&E) stain. It discusses why staining is necessary, common terminology used, theories of staining, types of stains and dyes, the roles of mordants and accentuators, and metachromasia. It then focuses on H&E stain, including the history and development of hematoxylin, the staining process, and different types of hematoxylin classifications based on the oxidation procedure and mordant used.
The document discusses the theory of staining in histology. It explains that staining allows tissues to be distinguished by providing different colors. Staining works through interactions between dyes and tissue components based on factors like charge, size, and chemical bonds. Differential staining is achieved through differences in binding capacity, rate, and strength between dyes and tissues. The document outlines various dye properties, classification systems, and staining methods. It also addresses factors influencing staining quality and provides examples of common problems and their solutions.
H and E staining is most important part of the histopathological diagnosis, this presentation is to highlight some important basic concept of the Staining.
This document provides an overview of hematoxylin and eosin staining. It discusses the theory behind staining, including how dyes interact with tissues through various bonding mechanisms. It also describes factors that influence staining results, such as rates of dye uptake and loss, binding site affinities, and tissue modification during fixation. The document highlights how hematoxylin and eosin work as the most commonly used routine stain in histopathology.
The document describes various staining techniques used to visualize microorganisms under a microscope. It discusses why staining is necessary given bacteria are transparent. It then covers different types of stains including simple, differential, special and vital stains. Various common stains are described like Gram stain, acid fast stain, Giemsa stain etc. Steps involved in staining like fixation, mordants, accentuation, progressive and regressive staining are explained.
The document provides information about Hematoxylin and Eosin staining. It states that H&E staining is widely used because of its simplicity, ability to clearly demonstrate tissue structures, and how it specifically stains cell nuclei blue-black and cytoplasm pink to red. Hematoxylin stains nuclei while Eosin stains cytoplasm. The document then discusses the chemistry and preparation of hematoxylin solutions, including how it is extracted from logwood and oxidized to its active form hematein. It also covers the use of mordants like aluminum, iron, and tungsten to improve hematoxylin's staining ability.
The Hematoxylin and Eosin stain (H&E) is the most widely used histological stain due to its comparative simplicity and ability to clearly demonstrate many different tissue structures. Hematoxylin stains cell nuclei blue-black and shows good intranuclear detail, while Eosin stains cell cytoplasm and connective tissue in varying shades of pink, orange, and red. The H&E stain outlines tissues and cellular components and is essential for identifying tissues and establishing the presence or absence of disease processes. Several hematoxylin solutions are described in the document, varying in their mordants, methods of oxidation, and intended uses in histology.
This document provides information on tissue processing and histopathologic techniques, focusing on staining. It defines staining as applying dyes to tissue sections to facilitate microscopic study. It then classifies stains based on pH, function, source, dye application technique, sequence, and color contrast. Specific staining techniques and commonly used stains are described for carbohydrates, lipids, proteins, and nucleic acids. Hematoxylin and eosin staining is explained as the most widely used staining procedure.
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The document discusses toluidine blue (TB) staining. It explains that TB is a metachromatic vital stain that selectively stains acidic tissue components. It outlines the structure, principle, and applications of TB staining, including its use in detecting potentially malignant oral lesions. The document also discusses the preparation, procedure, interpretation and sensitivity/specificity of TB staining.
1. Hematoxylin and eosin staining is the most widely used staining technique in histopathology. Hematoxylin stains nuclei blue/black while eosin stains cytoplasm and extracellular components pink.
2. Hematoxylin requires oxidation to produce hematein, the active dye, and uses a mordant like aluminum or iron salts to bind it to tissue. Eosin is a xanthine dye that stains cytoplasm and extracellular components red.
3. The basic steps of hematoxylin and eosin staining involve staining with hematoxylin, differentiating, bluing, staining with eosin, dehydration and mounting. Proper timing is needed for each step to achieve optimal
This document discusses staining techniques used to visualize bacteria under a microscope. It begins by explaining why staining is necessary given that bacteria are colorless and microscopic. It then describes different types of stains including basic stains that directly stain bacteria and acidic stains used for negative staining of the background. Key differential staining techniques are also summarized, including Gram staining which separates bacteria into Gram-positive and Gram-negative groups based on cell wall structure, and acid-fast staining used to identify Mycobacterium species. The document provides details on staining methods, the mechanisms of different staining techniques, and their importance in classifying and identifying bacterial specimens.
Dental Anatomy and Dental Histology Project on Hematoxylin & Eosin Stain - 1S...deepupadhyaya
The document discusses the Hematoxylin and Eosin staining technique. It begins with the presenter's information and acknowledgments. Hematoxylin and Eosin staining is then introduced as the most widely used histological staining technique. It allows for clear demonstration of numerous tissue structures as hematoxylin stains nuclei blue and eosin stains cytoplasm and connective tissues pink. The document proceeds to describe the individual stains hematoxylin and eosin in depth, including their properties and types. It concludes with an overview of the hematoxylin and eosin staining procedure for paraffin sections and cytology smears.
This document discusses the use of fluorescence dyes to stain cells and make their structures visible under a microscope. It explains that while early dyes stained broad cellular components, fluorescence dyes have increased specificity by binding to particular proteins or molecules. Common fluorescence dyes excite at specific wavelengths and emit light of longer wavelengths, allowing structures to be seen against a dark background. The document outlines the basic components of a fluorescence microscope and how it works, and notes some applications of fluorescence staining like detecting vitamins, drugs, and lipofuscin deposits in aging cells.
This document discusses various staining techniques used to visualize cells and internal structures. It describes the basic components and principles of dyes and stains, including chromophores, auxochromes and benzene rings. It outlines different types of stains categorized by molecular structure and charge. Basic techniques like simple staining, gram staining and acid-fast staining are explained in detail. The document compares properties of gram-positive and gram-negative cell walls. It provides examples of structures that are acid-fast and the importance of Ziehl-Neelsen staining for detecting Mycobacterium tuberculosis bacilli.
This document discusses various staining techniques used to visualize cells and internal structures. It describes the basic components and principles of dyes and stains, including chromophores, auxochromes and benzene rings. It outlines different types of stains categorized by molecular structure and electric charge. Basic techniques like simple staining, gram staining and acid-fast staining are explained in detail. The document compares properties of gram-positive and gram-negative cell walls. It provides examples of structures that are acid-fast and the importance of Ziehl-Neelsen staining for detecting Mycobacterium tuberculosis bacilli.
This document discusses Toluidine Blue staining, which is a metachromatic stain used to stain mast cell granules. Metachromasia is the property of certain stains to take on a color different from their original solution when bound to tissue components. Toluidine Blue is a basic, cationic dye that binds to acidic polysaccharides in tissues, polymerizing and changing color from blue to purple or red. The staining procedure involves dewaxing and hydrating tissue sections, staining with Toluidine Blue for 30 minutes, differentiating in alcohol, and mounting for microscopy. Mast cell granules, cartilage matrix, and mucopolysaccharides will be stained purple-red.
Tissue processing involves fixing, dehydrating, clearing, infiltrating, and embedding tissue samples to produce diagnostic microscope slides. It stabilizes tissues and removes water, replacing it with paraffin wax. Efficient agitation, heat up to 45°C, low viscosity reagents, and vacuum up to 50.79 kPa can reduce processing time. Stages include fixation with formalin, dehydration using graded alcohols, clearing with xylene or toluene, infiltration with paraffin wax, and embedding for microtomy. Proper orientation during embedding is important for diagnosis.
Special stains in dermato pathology - final copyariva zhagan
This document provides an overview of various special stains used in dermatopathology. It begins by explaining what stains are and their uses, such as enhancing contrast and examining tissues. It then describes the major categories of stains including those for carbohydrates, lipids, microorganisms, connective tissues, and minerals. Specific stains are also outlined, like PAS for glycogen and fungi, Masson's Trichrome for collagen, and Gram staining for bacteria identification. The document concludes by stating several stains used for visualizing structures like mast cells, amyloid, and various microbes under the microscope.
Connective tissue supports and connects organs and consists mainly of collagen, elastic fibers, and reticular fibers. Special staining techniques are used to identify these fibers, such as Masson's Trichrome to stain collagen blue and cytoplasm red, Gomori's to stain reticular fibers black, and Verhoeff's Van Gieson to stain elastic fibers blue-black. Connective tissue contains fibroblasts that produce fibers, as well as fat cells, mast cells, macrophages, and plasma cells. Muscle tissue includes skeletal striated muscle that is voluntary, cardiac muscle that is striated but involuntary, and smooth muscle.
In this humorous and data-heavy session, join us in a joyous celebration of life honoring the long list of SEO tactics and concepts we lost this year. Remember fondly the beautiful time you shared with defunct ideas like link building, keyword cannibalization, search volume as a value indicator, and even our most cherished of friends: the funnel. Make peace with their loss as you embrace a new paradigm for organic content: Pillar-Based Marketing. Along the way, discover that the results that old SEO and all its trappings brought you weren’t really very good at all, actually.
In this respectful and life-affirming service—erm, session—join Ryan Brock (Chief Solution Officer at DemandJump and author of Pillar-Based Marketing: A Data-Driven Methodology for SEO and Content that Actually Works) and leave with:
• Clear and compelling evidence that most legacy SEO metrics and tactics have slim to no impact on SEO outcomes
• A major mindset shift that eliminates most of the metrics and tactics associated with SEO in favor of a single metric that defines and drives organic ranking success
• Practical, step-by-step methodology for choosing SEO pillar topics and publishing content quickly that ranks fast
Did you know that while 50% of content on the internet is in English, English only makes up 26% of the world’s spoken language? And yet 87% of customers won’t buy from an English only website.
Uncover the immense potential of communicating with customers in their own language and learn how translation holds the key to unlocking global growth. Join Smartling CEO, Bryan Murphy, as he reveals how translation software can streamline the translation process and seamlessly integrate into your martech stack for optimal efficiency. And that's not all – he’ll also share some inspiring success stories and practical tips that will turbocharge your multilingual marketing efforts!
Key takeaways:
1. The growth potential of reaching customers in their native language
2. Tips to streamline translation with software and integrations to your tech stack
3. Success stories from companies that have increased lead generation, doubled revenue, and more with translation
We’ve entered a new era in digital. Search and AI are colliding, in more ways than one. And they all have major implications for marketers.
• SEOs now use AI to optimize content.
• Google now uses AI to generate answers.
• Users are skipping search completely. They can now use AI to get answers. So AI has changed everything …or maybe not. Our audience hasn’t changed. Their information needs haven’t changed. Their perception of quality hasn’t changed. In reality, the most important things haven’t changed at all. In this session, you’ll learn the impact of AI. And you’ll learn ways that AI can make us better at the classic challenges: getting discovered, connecting through content and staying top of mind with the people who matter most. We’ll use timely tools to rebuild timeless foundations. We’ll do better basics, but with the most advanced techniques. Andy will share a set of frameworks, prompts and techniques for better digital basics, using the latest tools of today. And in the end, Andy will consider - in a brief glimpse - what might be the biggest change of all, and how to expand your footprint in the new digital landscape.
Key Takeaways:
How to use AI to optimize your content
How to find topics that algorithms love
How to get AI to mention your content and your brand
The Secret to Engaging Modern Consumers: Journey Mapping and Personalization
In today's digital landscape, understanding the customer's journey and delivering personalized experiences are paramount. This masterclass delves into the art of consumer journey mapping, a powerful technique that visualizes the entire customer experience across touchpoints. Attendees will learn how to create detailed journey maps, identify pain points, and uncover opportunities for optimization. The presentation also explores personalization strategies that leverage data and technology to tailor content, products, and experiences to individual customers. From real-time personalization to predictive analytics, attendees will gain insights into cutting-edge approaches that drive engagement and loyalty.
Key Takeaways:
Current consumer landscape; Steps to mapping an effective consumer journey; Understanding the value of personalization; Integrating mapping and personalization for success; Brands that are getting It right!; Best Practices; Future Trends
In the face of the news of Google beginning to remove cookies from Chrome (30m users at the time of writing), there’s no longer time for marketers to throw their hands up and say “I didn’t know” or “They won’t go through with it”. Reality check - it has already begun - the time to take action is now. The good news is that there are solutions available and ready for adoption… but for many the race to catch up to the modern internet risks being a messy, confusing scramble to get back to "normal"
Build marketing products across the customer journey to grow your business and build a relationship with your customer. For example you can build graders, calculators, quizzes, recommendations, chatbots or AR apps. Things like Hubspot's free marketing grader, Moz's site analyzer, VenturePact's mobile app cost calculator, new york times's dialect quiz, Ikea's AR app, L'Oreal's AR app and Nike's fitness apps. All of these examples are free tools that help drive engagement with your brand, build an audience and generate leads for your core business by adding value to a customer during a micro-moment.
Key Takeaways:
Learn how to use specific GPTs to help you Learn how to build your own marketing tools
Generate marketing ideas for your business How to think through and use AI in marketing
How AI changes the marketing game
Everyone knows the power of stories, but when asked to come up with them, we struggle. Either we second guess ourselves as to the story's relevance, or we just come up blank and can't think of any. Unlocking Everyday Narratives: The Power of Storytelling in Marketing will teach you how to recognize stories in the moment and to recall forgotten moments that your audience needs to hear.
Key Takeaways:
Understand Why Personal Stories Connect Better
How To Remember Forgotten Stories
How To Use Customer Experiences As Stories For Your Brand
Yes, It's Your Fault Book Launch WebinarDemandbase
From Blame to Gain: Achieving Sales and Marketing Alignment to Drive B2B Growth.
Tired of the perpetual tug-of-war between your sales and marketing teams? Come hear Demandbase Chief Marketing Officer, Kelly Hopping and Chief Sales Officer, John Eitel discuss key insights from their new book, “Yes, It’s Your Fault! From Blame to Gain: Achieving Sales and Marketing Alignment to Drive B2B Growth.”
They’ll share their no-nonsense approach to bridging the sales and marketing divide to drive true collaboration — once and for all.
In this webinar, you’ll discover:
The underlying dynamics fueling sales and marketing misalignment
How to implement practical solutions without disrupting day-to-day operations
How to cultivate a culture of collaboration and unity for long-term success
How to align on metrics that matter
Why it’s essential to break down technology and data silos
How ABM can be a powerful unifier
Mastering Local SEO for Service Businesses in the AI Era is tailored specifically for local service providers like plumbers, dentists, and others seeking to dominate their local search landscape. This session delves into leveraging AI advancements to enhance your online visibility and search rankings through the Content Factory model, designed for creating high-impact, SEO-driven content. Discover the Dollar-a-Day advertising strategy, a cost-effective approach to boost your local SEO efforts and attract more customers with minimal investment. Gain practical insights on optimizing your online presence to meet the specific needs of local service seekers, ensuring your business not only appears but stands out in local searches. This concise, action-oriented workshop is your roadmap to navigating the complexities of digital marketing in the AI age, driving more leads, conversions, and ultimately, success for your local service business.
Key Takeaways:
Embrace AI for Local SEO: Learn to harness the power of AI technologies to optimize your website and content for local search. Understand the pivotal role AI plays in analyzing search trends and consumer behavior, enabling you to tailor your SEO strategies to meet the specific demands of your target local audience. Leverage the Content Factory Model: Discover the step-by-step process of creating SEO-optimized content at scale. This approach ensures a steady stream of high-quality content that engages local customers and boosts your search rankings. Get an action guide on implementing this model, complete with templates and scheduling strategies to maintain a consistent online presence. Maximize ROI with Dollar-a-Day Advertising: Dive into the cost-effective Dollar-a-Day advertising strategy that amplifies your visibility in local searches without breaking the bank. Learn how to strategically allocate your budget across platforms to target potential local customers effectively. The session includes an action guide on setting up, monitoring, and optimizing your ad campaigns to ensure maximum impact with minimal investment.
Google Ads Vs Social Media Ads-A comparative analysisakashrawdot
Explore the differences, advantages, and strategies of using Google Ads vs Social Media Ads for online advertising. This presentation will provide insights into how each platform operates, their unique features, and how they can be leveraged to achieve marketing goals.
In today's digital world, customers are just a click away. "Grow Your Business Online: Introduction to Digital Marketing" dives into the exciting world of digital marketing, equipping you with the tools and strategies to reach new audiences, expand your reach, and ultimately grow your business.
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Breaking Silos To Break Bank: Shattering The Divide Between Search And SocialNavah Hopkins
At Mozcon 2024 I shared this deck on bridging the divide between search and social. We began by acknowledging that search-first marketers are used to different rules of engagement than social marketers. We also looked at how both channels treat creative, audiences, bidding/budgeting, and AI. We finished by going through how they can win together including UTM audits, harvesting comments from both to inform creative, and allowing for non-login forums to be part of your marketing strategy.
I themed this deck using Baldur's Gate 3 characters: Gale as Search and Astarion as Social
Lily Ray - Optimize the Forest, Not the Trees: Move Beyond SEO Checklist - Mo...Amsive
Lily Ray, Vice President of SEO Strategy & Research at Amsive, explores optimizing strategies for sustainable growth and explores the impact of AI on the SEO landscape.
The Strategic Impact of Storytelling in the Age of AI
In the grand tapestry of marketing, where algorithms analyze data and artificial intelligence predicts trends, one essential thread remains constant — the timeless art of storytelling. As we stand on the precipice of a new era driven by AI, join me in unraveling the narrative alchemy that transforms brands from mere entities into captivating tales that resonate across the digital landscape. In this exploration, we will discover how, in the face of advancing technology, the human touch of a well-crafted story becomes not just a marketing tool but the very essence that breathes life into brands and forges lasting connections with our audience.
As the call for for skilled experts continues to develop, investing in quality education and education from a reputable https://www.safalta.com/online-digital-marketing/best-digital-marketing-institute-in-noida Digital advertising institute in Noida can lead to a a success career on this eve
2. INTRODUCTIONINTRODUCTION
• If something have color that helps to know things,
differentiate & express , particular entity
• Dyes are the agents which are get absorb into on which
it applied and resist to soap washing and sunlight
• Colors are agent which only adsorb on surface and
dose not resist to sunlight and soap
• “Biological dyes” - highlight and differentiate
tissue components and allow them to be seen under
the microscope
3. • The tissue section is colorless because the fixed protein
has the same refractive index as that of glass
• The sections,, are colorless and different components
can’t be appreciated
The Need to Stain
• Outlines tissues and cellular components.
• Identification of tissues.
• Establishes the presence or absence of
disease processes.
4. • Staining them by different coloured dyes, having
affinities of specific components of tissues, makes
identification and study of their morphology possible.
• stains give a contrast to the microscopic slide.
5.
6. • Stain is the reagent used to generate the color
• Staining : process of coloring cells, cellular constituent
& tissue fibers to facilitate optical differentiation by
microscopic examination.
• Is the union between a colored dye & a tissue substrate
which resists simple washing
7.
8. NATURAL DYESNATURAL DYES
• Dyes derived from plants, invertebrates or minerals.
• Majority natural dyes are derived from vegetables
1.Cochineal
2.Orcien
3.Saffron
4.Haematoxylin
9. 1. Cochineal1. Cochineal
• Source from dried female insect : Coccus cacti
• Cochineal dye was used by the Aztec and Maya peoples
of Central and North America
• Synonyms : cochineal extract", "carmine", "crimson
lake", "natural red 4", "C.I. 75470", "E120“
• Biological Morphology :Three pairs of legs and seven
pairs of antenna
11. • During egg development stage insects are collected.
• They are killed by water boiling or stove burning it gives
purplish black colors.
• When they are killed by charcoal fumes or burning with
sulphar it develops purplish grey color.
• Chemical constituent : 10% anthorquinone dye known
as carminic acid which brilliant purple.
12. • Carmine was introduced in 1849 by Goppert
and Cohn.
13. 2.2. OrceinOrcein
• Derived from lichens
• Demonstrate elastic fibers and, following permanganate
oxidation (Shikata's modification), hepatitis B surface
antigen, copper associated protein, and sulfated mucins
14. 3.3. SaffronSaffron
• From the dried stigmata of Crocus sativus
flower("saffron crocus“)
• In 1714 Leeuwenhoek employed saffron on sections of
muscle fibres
• Coloring substance is termed as “ crocin “
15. Synthetic DyesSynthetic Dyes
• Alizarin in 19th century -1st synthetic dye
• 1.Acidic dyes
• Dyes that have affinity towards basic part of tissue
components
• Acids have a negative charge in an electric field, they
migrate towards anode (anions)
• Tissue having a positive charge will attract with dyes with
a negative charges
• Such tissues are acidophilic
• Eg: Orange G, Eosine, Nigrosine
16. 2.Basic dyes
• Dyes have affinity towards acid part of tissues
• Bases have a positive charge ,migrate towards Cathode
(cations )
• Tissue carrying negative charge will attract dyes of
positive charge
• These tissue are basophilic
• Eg: Methylene blue, Crystal violet,
Basic fuchsin,Malachite green,
Hematoxylin
17. • 3.Neutral Dyes
• Compound stains
• Produced by neutralizing suitable acid dyes with suitable
Basic dyes
• When aqueous solutions of two dyes brought together ,an
interchange of ions takes place
• Precipitate is formed which is a two color- dye
• It is insoluble in pure water but dissolve in water
containing excess of acid/basic dye from which they are
formed
• Eg: Romanowsky stains
18. • Sections stained with acidic or basic dyes are mounted in
non aqueous media to prevent loss of dye
19. Theory of staining
• 1.Physical theory
• Solubility: stain is more soluble in fat than in the solvent
• Adsorption :large body attracts minute particles from the
surrounding medium
20. • Chemical theory
• Basic structure of all the stains is Benzene
• Benzene has a ring structure, it give flexibility
• Chromophore :Chromophore :Chemical group introduced into benzene
ring by substituting Hydrogen
• Benzene + chromophoreBenzene + chromophore ChromogenChromogen
21. • Along with chromogen it is necessary to introduce
Ionizing groups-Auxochromes
• After the addition of Auxochromes ,colored compound
stains the tissue and be resistant to simple washing
• STAIN = AuxochromeAuxochrome+ChromogenChromogen
22. Principle of staining
1.Electrostatic Bonding1.Electrostatic Bonding
•Affinity between Opp.ionic groups of dye and tissue
2.Hydrogen Bonding2.Hydrogen Bonding
•Form of dye tissue attraction
•Arises when hydrogen atom lies between two
electronegative atoms
•Weak bonds, occur readily in water, Occur between dye
and water
23. • 3.Van der waal forces3.Van der waal forces
• Occur between reagent and tissue substrates
• Involves various intermolecular attractions
Dipole-dipole
Dipole induced dipole
Dispersion forces
• These forces are polar attractions
• Effective over a short distance
• Eg : Staining of elastic fibres by orcein
24. • 4.Covalent Bonding4.Covalent Bonding
• Can occur between the stain and the tissue
• Covalent bonding involves sharing of electrons
• Sharing of 2,3 or more electrons between 2 atoms –
Multiple covalent bonds
• This bonding is of significance in mordant dying process
25. MordantsMordants
• “To bite”
• A polyvalent metal ion which forms coordination
complexes with certain dyes
• A substance which acts as an intermediary between dye
and tissue, thus increasing the affinity between them
• Strictly applicable to salts and hydroxides of divalent
and trivalent metals
• A mordant is a metal with a valency of at least two
• Commonly used in histotechnology are aluminum and
ferric iron (Valencies 3 )
26. PRINCIPLE OF MORDANT
• Hematin is anionic.
• Tissue is also anionic.
• Therefore hematin has poor affinity for tissue
• Making hematin adequate as a nuclear stain with the
presence of a 3rd element (mordant)
• Mordant forms a link between the “tissue and the stain”
27. • The chelate formed from a mordant dye and a metal is
called a lake
28. Mordant-Dye application
• Mordant is applied first, followed by the dye.
e.g Heidenhain’s iron hematoxylin
• Mordant and dye are mixed together and then applied.
Commonly done in histotechnology
e.g Alum hematoxylin solutions
• Dye applied first, followed by the mordant.
Hardly done in histotechnology
29. AccentuatorsAccentuators
• Substances which increase the staining intensity ,
crispness and selectivity of dye.
• Eg : KOH in Lofflers methylene blue
phenol in carbol fuschin & carbol thionin
Aniline dye in gentian violet
• They do not form lakes with dyes
• They act by changing pH
30.
31. AcceleratorsAccelerators
• Accentuators used in metallic impregnation technique for
the nervous system.
• e.g chloral hydrate, Barbital
Trapping agentsTrapping agents
• Agents which inhibit removal of dyes from tissue
• e.g Iodine in Gram stain
NaCl in Gram Weigert method
32.
33. RipeningRipening
• Oxidation of haematoxylin
• This process converts the haematoxylin to haematin
1.Natural
• Carried out by exposure to light and air
• Slow process – takes about 3-4 months
• Resultant solutions seem to retain its staining ability for a
long time
• e.g Ehrlich’s & Delafield’s haematoxylin solutions
34. 2.Chemical oxidation
• Oxidation occurs instantaneously
• These solutions have a shorter shelf life
• Oxidation using sodium iodate – most commonly used
e.g. Mayer’s hematoxylin
• Oxidation by mercuric oxide e.g. Harris’s hematoxylin
• Ferric chloride and potassium permanganate can also used
35. Staining ReactionsStaining Reactions
• Absorption or direct staining – tissue penetrated by
dye solution.
• Indirect staining – using intermediate treatment with
mordant
• Physical staining – simple solubility of dye in element
of tissue.
• Chemical staining – formation of new substance e.g.
PAS
• Adsorption phenomenon – accumulation on the surface
of the compound.
36. Progressive stainProgressive stain
• Tissue is left in the stain just enough to reach the
endpoint
• Frequent monitoring of stain quality may be needed to
determine when staining is complete
• The staining intensity is controlled by the time it is
immersed in the solution
• Eg : progressive staining using Gill’s Hematoxylin
37. Regressive StainingRegressive Staining
• Involves over-staining where the dye completely
saturates all tissue elements
• The tissue is then selectively de-stained using a process
called differentiation
• Eg :Harris Hematoxylin is popularly used
regressively in many histology labs for routine H & E
staining
• Sharper degree of staining is obtained
38. DiffrentiationDiffrentiation
• Selective De-staining
• Removal or washing out of excess stain until the color is
retained only by the tissue component that is to be
studied
• Differentiation is achieved by using a dilute acid, most
typically 1% acid alcohol
• Differentiation is halted by washing in water when the
desired endpoint is reached
• Exposure to air may oxidize and improve the process.
39. BlueingBlueing
• Blueing is the process of shifting the color
from reddish to purple blue by the application of
a weak alkaline solution
• Blueing is utilized in both progressive and
regressive techniques
• Eg :Tap water , Scott’s tap water(Magnesium
sulfate ,Sodium bicarbonate )Saturated Lithium
carbonate, Ammonia in distilled water
40.
41. MetachromasiaMetachromasia
• Dye has the ability to change its color without changing
chemical structure
• Eg : Thiazine group like thionin,toludine blue,Azure A,B
• When negatively charged groups on the tissue react with
cationic dyes
• Enhanced when intermolecular distance reduced
42.
43. • Polychromasia:Polychromasia: Exhibit two or more shades of color
reactions Eg : Leishman,Giemsa
• Orthochromasia :Orthochromasia : Color of the dye doesn’t change
44. History of hematoxylin
• It was independently introduced in 1865 and 1875, by
Böhmer and Fischer respectively.
• Primary diagnostic technique in the histo-pathology
laboratory.
• Waldeyer established the use of
hematoxylin in histology in 1862.
45. Cont…
• Bohmer combines hematoxylin with
alum as a mordant & obtained more
specific staining in 1864.
• Heindenhan introduced classical Iron -
Alum hematoxylin method
• Ehrlich overcame the instability of alum
hematoxylin by adding glacial acetic acid.
46. HEMATOXYLIN
• The word hematoxylin is derived from old Greek word
Haimato (blood) & Xylon (wood) , referring to its dark red
colour in natural state.
• A natural dye extracted from the log wood of the tree
Haematoxylon campechianum, mainly seen in Campeche
state of Mexico & also available in West Indies
47. Extraction
• Small pieces of the log wood is boiled in water
• First orange- red solution is formed.
• On cooling it turn into black solution.
• Then precipitated with urea or ether.
• Purified and sold commercially.
48. • Hematoxylin itself is not a stain
• On oxidation it produces hematein (poor dye).
• Commonly used dye in histopathology, cytology,
immuno histochemistry.
• Best nuclear stain.
• Basic in nature, stains acidic component of the
tissue, nucleus, mitochondria etc.
49. oxidation
• Oxidation of hematoxylin is called RIPENING.
• Hematein can be produced in 2 ways
i. Natural ripening
ii. Chemical ripening
50. Cont…
Natural ripening
• By exposure to sunlight & air
• Slow process, takes approximately 3 months.
• Staining life of the dye is longer.
Chemical ripening
• Chemical agents converts hematoxylin to hematein
instantly.
• Sodium iodate , mercuric oxide commonly used, Ferric
chloride and potassium permanganate can also be used.
• Short useful lifespan
52. MORDANT
• Hematein is a weak basic dye having a poor affinity for
tissue
• Mordant is a chemical substance that increases the
affinity of dye for tissue and its staining efficiency by
forming link between the stain and the tissue.
• It’s a metal with valence of atleast two.
• Various mordants used are aluminium ,iron, lead,
phosphotungstic acid etc.
53. Types of hematoxylin
According to the mordant used
Alum hematoxylin
Iron hematoxylin
Tungsten hematoxylin
Molybdenum hematoxylin
Lead hematoxylin
Hematoxylin with out mordant
54. ALUM HEMATOXYLIN
• Mordant used are aluminum salts, either aluminum
potassium sulphate (potash alum) or aluminum
ammonium sulphate (ammonium alum).
• Mainly used in routine H and E staining.
• Has different types but all of them stain the nuclei blue-
black.
• These are used when counter stain does not contain an
acid.
• Can be used progressively or regressively.
55. 1. Ehrlich’s hematoxylin
• Strong stain for nuclei, stains intensely & crisply
• Oxidized naturally
• Stained sections fade slowly.
• Not ideal for frozen sections
e
56. 2. Harri’s hematoxylin
• Widely used in exfoliative cytology as a nuclear
stain
• Chemical oxidation – mercuric oxide
• Life span is short : 2-3 months
57. 3. Mayer’s hematoxylin
• Used as anuclear counterstain where the cytoplasmic
material needed to demonstrate.
• Used as a progressive stain
• Chemical oxidation – sodium iodate
• Gives little or no staining of mucopolysaccharide
material.
60. 6. Carazzi’s hematoxylin
• Chemically ripened with potassium iodate
• Used as a progressive nuclear stain.
• Largely confined to use with frozen sections
61.
62. Disadvantage of alum hematoxylin
• Alum hematoxylin nuclear stain is sensitive
to the subsequently applied acidic solutions.
• Common examples are Van Gieson & trichrome
stain.
• Satisfactory staining can be achieved by using Iron
mordanted hematoxylin, which resist effect of picric
acid.
• Combination of celestine blue with an alum
63. Celestine blue
• Oxazine Dye
• Has little useful coloring property of its own
• It forms an additional strong mordant with certain
hematoxylins.
• Used as a preliminary to alum hematoxylin staining.
• Resistant to the effects of acid.
• Ferric salt in the celestine blue solution strengthens the
bond between the nucleus and the alum hematoxylin to
provide a strong nuclear stain which is reasonably
resistant to acid.
64. Preparation
• Celestine blue B : 2.5g
• Ferric ammonium suphate : 25g
• Glycerine : 70ml
• Distilled water : 500ml
Ferric ammonium sulphate is dissolved in cold distilled water with
stirring.
The celestine blue B is added to this solution and the mixture is
boiled for few minutes.
Filtered
Glycerine is added, filter
65. Iron hematoxylin
• Iron salts used both as mordant & oxidizing agent
• Commonly used salts are ferric chloride & ferric
ammonium sulphate.
• Over oxidation is the problem : to avoid this prepare
mordant/ oxidant & hematoxylin seperately & mix them
immediately before use.
66. Stain Mordant oxidation Application/
results
Staining time
Weigert’s
hematoxylin
Ferric chloride Natural Nuclear stain
with acid dye,
Stains nucleus
brown to black
15-30 min
Heidenhains’s
hematoxylin
Ferric
ammonium
sulphate
Natural mitochondria,
chromatin &
muscle fiber
striation stains
black or dark
grey- black
30-45 min at
60º C
12- 24 hrs at
RT
Verhoeff’s
hematoxylin
Ferric chloride Natural Stains elastic
fibers as black
25- 60 min
Loyez
hematoxylin
Ferric
ammonium
sulphate
Natural Myelin
67. Tungsten hematoxylin
• Mallory phosphotungstic acid hematoxylin (PTAH) is an
example
• Phosphotungstic acid is used as mordant.
• Possible to prepare a staining solution using hematein ;
instead of hematoxylin. Oxidation not required, can be
used immediately , but short lived.
68. • Can be naturally oxidized, takes months to ripen: usable
for many years
• Can be oxidized chemically using potassium
permanganate.
• Applicable to nervous tissue & also used to stain
muscle striations and fibers
69. Molybdenum hematoxylin
• Molybdic acid as the mordant.
• For the demonstration of collagen, coarse reticulin&
granules in endocrine cells.
• Hydrogen per oxide is used for oxidation.
Lead hematoxylin
• Lead salts act as the mordant.
• Identification of endocrine cells in some in tumors.
71. Eosin
• Most suitable stain to combine with alum hematoxylin.
• Eosins are xanthine dyes (tetrabromofluorescein)
• TYPES OF EOSIN - commercially available
Eosin Y
Ethyl eosin
Eosin B
72. Cont…
o Eosin Y
• Eosin yellowish
• Most widely used
• It is water & alcohol soluble.
• Used as a cytoplasmic stain - 0.5-1% solution in distilled
water with a Crystal of thymol - prevent fungal growth.
• Addition of Acetic acid sharpens the staining
o Ethyl eosin (eosin alcohol-soluble)
o Eosin B (eosin bluish, erythrosine B)
73. Cont…
DIFFERENTIATION OF EOSIN
• Occurs in the subsequent tap water wash
• Further differentiation occurs during the dehydration
through the alcohols
ALTERNATIVES FOR EOSIN
Phloxine
Bierbrich scarlet
saffranine
76. Troubleshooting in H&E staining
Problems Possible causes Remedies
Pale stained nuclei 1. Too much differentiation
2. Too less time in
haematoxylin
3. Due to excessive
decalcification
4. Haematoxylin is over
oxidized
1. Stain in haematoxylin
again
2. Keep in haematoxylin for
longer duration
3. Not possible to correct
4. Change the haematoxylin
solution
Darkly stained nuclei 1. Too short differentiation
2. Too much time in
haematoxylin
3. Thick section
1. Decolorize and do
optimum differentiation
2. Decolorize and give
appropriate time in
haematoxylin
3. Recut thin section
Nuclei looks reddish brown 1. Insufficient bluing
2. Haematoxylin is
degenerating
1. Restain by giving more
time in bluing step
2. Check the oxidation status
of haematoxylin
77. Problems Possible causes Remedies
Pale coloured cytoplasm
by eosin
1. Too thin section
2. The eosin solution has
pH more than 5
3. Too much dehydration
of
the section in alcohol
1. Recut the section
properly
2. May be due to dilution
of
eosin by the carryover
bluing solution. Check
pH of eosin solution
3. Do not keep the slide in
alcohol for a long time
Bluish-black precipitate May be due to precipitation
of haematoxylin
Filter the haematoxylin
staining solution
Staining is irregular and
spotty
Improper deparaffinization Keep the slide in xylene for
longer time to remove
the paraffin
Dark-blue stain at
the edge of the
tissue sections
Due to heating artefact for
using electrocautery
No solution
Water bubbles in the
section
Incomplete dehydration Remove the mounting
medium and coverslip.
Keep the section in
absolute alcohol for