- 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 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.
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.
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 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 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 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.
This document discusses various special stains used in pathology and their principles and results. It describes hematoxylin and eosin staining which stains cell nuclei purple and cytoplasm pink. It also discusses mucin stains like PAS, mucicarmine and Alcian blue; melanin, lipochrome, iron, fat, AFB, fungal and connective tissue stains. It provides examples of stained tissues and conditions and includes an assignment with questions.
This document discusses fixatives used in histopathology. It describes the process of fixation and how fixatives preserve tissue by denaturing or precipitating proteins. The ideal properties of a fixative are described, including preventing autolysis and allowing for staining. Common fixatives are classified and their mechanisms and uses are explained. Factors that affect fixation such as temperature, size, volume ratio, time, choice of fixative, and penetration are also summarized.
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.
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.
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 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 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 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.
This document discusses various special stains used in pathology and their principles and results. It describes hematoxylin and eosin staining which stains cell nuclei purple and cytoplasm pink. It also discusses mucin stains like PAS, mucicarmine and Alcian blue; melanin, lipochrome, iron, fat, AFB, fungal and connective tissue stains. It provides examples of stained tissues and conditions and includes an assignment with questions.
This document discusses fixatives used in histopathology. It describes the process of fixation and how fixatives preserve tissue by denaturing or precipitating proteins. The ideal properties of a fixative are described, including preventing autolysis and allowing for staining. Common fixatives are classified and their mechanisms and uses are explained. Factors that affect fixation such as temperature, size, volume ratio, time, choice of fixative, and penetration are also summarized.
Hematoxylin and eosin (H&E) staining is the most common histology stain. Hematoxylin stains cell nuclei blue by binding to DNA and RNA, while eosin stains cytoplasm and extracellular components pink. The staining process involves deparaffinizing tissue sections, staining with hematoxylin, differentiating with acid to remove excess stain, staining with eosin, and mounting for examination. Hematoxylin is extracted from logwood and oxidized to hematin, which binds tissue as a cationic dye with a mordant like alum. Eosin Y is the typical counterstain used to visualize cytoplasm. Together, H&E staining provides excellent contrast to study cell and
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
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
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.
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.
Hematoxylin and eosin staining is the most widely used histological staining technique. It uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The process involves dewaxing tissue sections, hydrating them, staining with hematoxylin, differentiating with acid alcohol, counterstaining with eosin, dehydrating, and mounting. Hematoxylin requires a mordant like aluminum or iron to stain tissues. Commonly used hematoxylins include Harris', Mayer's, Ehrlich's, and Delafield'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 PAS stain identifies polysaccharides, mucus substances, basement membranes, and some fungi by causing them to appear magenta under the microscope. It works by first using periodic acid to oxidize carbohydrate groups, then exposing the tissue to Schiff's reagent, which causes aldehyde groups produced in the first step to appear magenta. The PAS stain is used to identify conditions involving abnormal glycogen storage or mucus production, such as certain tumors, infections, and genetic diseases. It helps diagnose issues in tissues from the liver, kidney, lung, muscle, and other organs.
The H&E stain is the most common stain used in histology. It involves staining tissue samples with hematoxylin, which stains nuclei blue, followed by a counterstain with eosin, which stains cytoplasm and other tissue structures pink. The staining process involves deparaffinization, hydration, hematoxylin staining, differentiation, bluing, eosin counterstaining, dehydration, clearing, and coverslipping. Both automated and manual methods can be used to perform the H&E stain, and quality control measures help ensure consistent, high-quality results.
Staining ( rouine and special in cytology) rajiv kumarrajusehrawat
The document discusses staining techniques used in histology and cytology. It provides details on the preparation, components, and use of common stains including Hematoxylin, Giemsa stain, Papanicolaou stain, and Periodic acid–Schiff stain. The stains are used to differentially color structures like nuclei, cytoplasm, muscles, bones, parasites and glycogen under the microscope to enable examination of tissue samples and identification of cells and microorganisms.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
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 describes the hematoxylin and eosin stain (H&E stain) procedure performed at a 125-bed community hospital. Over a 30-day period, 291 specimens were analyzed, with 15 found to contain cancerous cells and 276 containing benign cells. The H&E stain involves hematoxylin staining cell nuclei blue-black and eosin staining cell cytoplasm and connective tissues shades of pink and red. This allows differentiation of tumor/cancer cells from normal cells under a microscope.
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.
The document discusses H&E (hematoxylin and eosin) staining, which is the most widely used staining technique in histopathology. H&E staining differentially colors tissue components, with hematoxylin staining nuclei blue and eosin staining cytoplasm and other tissues shades of pink. The process involves deparaffinizing tissue sections, staining with hematoxylin, differentiating with acid, counterstaining with eosin, and mounting for examination under a microscope. H&E staining allows clear visualization and analysis of cells and structures to enable histopathological diagnosis.
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 provides information on various histological staining techniques used to identify different types of tissues and biomolecules. It discusses connective tissue stains like Van Gieson's stain, Masson's trichrome stain, and Verhoeff's stain used to identify collagen fibers. It also describes reticulin stains, elastic stains, carbohydrate stains like periodic acid Schiff, and mucin stains like Alcian blue to identify different components of tissues under the microscope. Procedures for each stain are outlined along with the expected results.
The document discusses mounting media used to embed specimens on microscope slides under a coverslip. There are two main categories of mounting media: resinous (organic) media, which are dissolved in solvents like xylene and harden through evaporation, and aqueous media for specimens mounted in water. The ideal mounting medium is transparent, colorless, protects specimens from damage, and has a refractive index close to that of glass. Common mounting media include Canada balsam, Euparal, glycerin jelly, and aqueous solutions. Proper mounting techniques help prevent air bubbles between the specimen and coverslip.
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 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.
Hematoxylin and eosin (H&E) staining is the most common histology stain. Hematoxylin stains cell nuclei blue by binding to DNA and RNA, while eosin stains cytoplasm and extracellular components pink. The staining process involves deparaffinizing tissue sections, staining with hematoxylin, differentiating with acid to remove excess stain, staining with eosin, and mounting for examination. Hematoxylin is extracted from logwood and oxidized to hematin, which binds tissue as a cationic dye with a mordant like alum. Eosin Y is the typical counterstain used to visualize cytoplasm. Together, H&E staining provides excellent contrast to study cell and
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
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
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.
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.
Hematoxylin and eosin staining is the most widely used histological staining technique. It uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The process involves dewaxing tissue sections, hydrating them, staining with hematoxylin, differentiating with acid alcohol, counterstaining with eosin, dehydrating, and mounting. Hematoxylin requires a mordant like aluminum or iron to stain tissues. Commonly used hematoxylins include Harris', Mayer's, Ehrlich's, and Delafield'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 PAS stain identifies polysaccharides, mucus substances, basement membranes, and some fungi by causing them to appear magenta under the microscope. It works by first using periodic acid to oxidize carbohydrate groups, then exposing the tissue to Schiff's reagent, which causes aldehyde groups produced in the first step to appear magenta. The PAS stain is used to identify conditions involving abnormal glycogen storage or mucus production, such as certain tumors, infections, and genetic diseases. It helps diagnose issues in tissues from the liver, kidney, lung, muscle, and other organs.
The H&E stain is the most common stain used in histology. It involves staining tissue samples with hematoxylin, which stains nuclei blue, followed by a counterstain with eosin, which stains cytoplasm and other tissue structures pink. The staining process involves deparaffinization, hydration, hematoxylin staining, differentiation, bluing, eosin counterstaining, dehydration, clearing, and coverslipping. Both automated and manual methods can be used to perform the H&E stain, and quality control measures help ensure consistent, high-quality results.
Staining ( rouine and special in cytology) rajiv kumarrajusehrawat
The document discusses staining techniques used in histology and cytology. It provides details on the preparation, components, and use of common stains including Hematoxylin, Giemsa stain, Papanicolaou stain, and Periodic acid–Schiff stain. The stains are used to differentially color structures like nuclei, cytoplasm, muscles, bones, parasites and glycogen under the microscope to enable examination of tissue samples and identification of cells and microorganisms.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
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 describes the hematoxylin and eosin stain (H&E stain) procedure performed at a 125-bed community hospital. Over a 30-day period, 291 specimens were analyzed, with 15 found to contain cancerous cells and 276 containing benign cells. The H&E stain involves hematoxylin staining cell nuclei blue-black and eosin staining cell cytoplasm and connective tissues shades of pink and red. This allows differentiation of tumor/cancer cells from normal cells under a microscope.
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.
The document discusses H&E (hematoxylin and eosin) staining, which is the most widely used staining technique in histopathology. H&E staining differentially colors tissue components, with hematoxylin staining nuclei blue and eosin staining cytoplasm and other tissues shades of pink. The process involves deparaffinizing tissue sections, staining with hematoxylin, differentiating with acid, counterstaining with eosin, and mounting for examination under a microscope. H&E staining allows clear visualization and analysis of cells and structures to enable histopathological diagnosis.
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 provides information on various histological staining techniques used to identify different types of tissues and biomolecules. It discusses connective tissue stains like Van Gieson's stain, Masson's trichrome stain, and Verhoeff's stain used to identify collagen fibers. It also describes reticulin stains, elastic stains, carbohydrate stains like periodic acid Schiff, and mucin stains like Alcian blue to identify different components of tissues under the microscope. Procedures for each stain are outlined along with the expected results.
The document discusses mounting media used to embed specimens on microscope slides under a coverslip. There are two main categories of mounting media: resinous (organic) media, which are dissolved in solvents like xylene and harden through evaporation, and aqueous media for specimens mounted in water. The ideal mounting medium is transparent, colorless, protects specimens from damage, and has a refractive index close to that of glass. Common mounting media include Canada balsam, Euparal, glycerin jelly, and aqueous solutions. Proper mounting techniques help prevent air bubbles between the specimen and coverslip.
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 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.
Haematoxylin and eosin (H&E) staining is the most widely used histological stain. It uses haematoxylin, which stains nuclei blue, and eosin, which stains cytoplasm and extracellular components varying shades of pink. There are different types of haematoxylin depending on the mordant used, including alum, iron, tungsten, and un-mordanted haematoxylins. Alum haematoxylins like Mayer's and Harris' are most commonly used in H&E staining. Iron haematoxylins yield a stronger nuclear stain and can demonstrate more structures. Understanding the different haematoxylins allows their more informative use in diagnostic pathology.
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.
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 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.
The H&E stain is commonly used in histology because it clearly demonstrates cell nuclei and cytoplasm. Hematoxylin stains nuclei blue-black while eosin stains cytoplasm and connective tissues shades of pink. Hematoxylin is extracted from logwood and oxidized to hematein, its active ingredient. Eosin is a xanthine dye that is yellow and stains cytoplasm. In the H&E staining process, tissues are stained with hematoxylin, differentiated with acid, and counterstained with eosin to visualize nuclei in blue and cytoplasm in pink. The H&E stain technique involves dewaxing, rehydrating, staining, differentiation, dehydration and mounting of tissue sections.
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.
Hematoxylin is a natural dye extracted from the heartwood of the bloodwood tree. It is converted to hematein during preparation of staining solutions, usually using an oxidizing agent. Hematoxylin and eosin staining is commonly used in histology, staining nuclei blue with hematoxylin and cytoplasm red with eosin. The dyes require a mordant like aluminum or iron to bind to tissues. Sections are deparaffinized, stained, dehydrated and mounted for examination under a microscope using an aqueous or resinous mounting medium.
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.
procedure for Staining on routine histopath.pptIvanaUngajan2
This document discusses hematoxylin and eosin (H&E) staining techniques. It describes the appearance of the nucleus and cytoplasm in H&E stained slides. Key components that stain with H&E are described such as the nucleolus, chromatin, ribosomes and endoplasmic reticulum. The mechanisms of nuclear and cytoplasmic staining are explained in relation to pH and the isoelectric point of proteins. Common nuclear and plasma stains used in H&E, including hematoxylin, hematein and eosin, are outlined with details on their preparation, use and staining properties.
This document discusses staining techniques used in histopathology. It describes the purpose of staining as outlining tissues and cellular components to identify tissues and establish the presence or absence of disease. The most common staining technique is hematoxylin and eosin (H&E) staining. Hematoxylin stains nuclei blue and eosin stains cytoplasm pink, allowing clear visualization of tissue structures. The document provides details on the preparation and use of hematoxylin and eosin stains as well as other specialized staining techniques.
Haematoxylin and Eosin Staining in EcotoxicologyAzisKemalFauzie
Hematoxylin is extracted from the logwood tree and has been used as a biological stain since the 1860s. It works by binding to DNA and RNA in cell nuclei, staining them purple. Several formulations exist including Mayer's hematoxylin, which is a progressive stain using hematoxylin, aluminum salts, and other compounds. Progressive stains take longer but allow more control over nuclear staining compared to faster regressive stains that require differentiation steps. Overall, hematoxylin continues to be widely used for histology due its ability to clearly highlight cell nuclei.
Staining involves using dyes to make tissues more visible under a microscope. Stains work by binding differently to various cell components based on their chemical properties. Organic stains have two key parts - a chromophoric group that gives the stain its color, and an auxochromic group that allows the stain to bind to cells. Some stains produce different colors than their actual hue, known as metachromasia. Common stains include hematoxylin, safranin O, fast green FCF, and carmine. Natural dyes were originally used but now synthetic stains from coal tar are more common.
This document discusses different types of pigments found in tissues. It describes endogenous pigments such as hematogenous pigments (hemosiderin, hemoglobin), bile pigments, porphyrins, melanin, lipofuscin and chromaffin. Hemosiderin contains stored iron and appears as yellow-brown granules. Various stains can demonstrate iron, including Perl's Prussian blue. Hemoglobin transports oxygen and is stained blue with patent blue. Bile pigments are breakdown products of hemoglobin transported to the liver and gallbladder. Lipofuscin is an oxidation product that accumulates with age in various tissues. The document also discusses exogenous pigments and methods for demonstrating various endogenous pigments
This document discusses Perls stain, which is used to identify iron deposits in tissue samples. It provides background on pigments in living tissue, including endogenous pigments like hemosiderin and hematogenous pigments. The history of Prussian blue and its use as Perls stain is described. The principle of the stain is that hydrochloric acid releases ferric ions from hemosiderin, which then react with potassium ferrocyanide to form insoluble Prussian blue pigment. Staining protocols, quality control, and clinical applications for identifying iron deposits in organs are covered.
The document discusses various staining methods used in histology. It describes how stains are classified as basic, acid or neutral dyes based on their chemical properties. Staining methods can be simple, compound, indirect, direct, progressive, regressive or selective depending on the technique. Commonly used stains include hematoxylin, eosin, safranin and Mallory's trichrome. The hematoxylin and eosin stain is also described in detail, outlining the staining process and factors that can influence results. Different types of mounting media are discussed for permanent or temporary coverslipping of tissue samples.
This document defines and describes emulsions. It states that an emulsion is an unstable mixture of two immiscible liquids stabilized by an emulsifying agent. Emulsions are classified as simple (macro) emulsions, multiple emulsions, or microemulsions. Simple emulsions can be oil-in-water or water-in-oil, while multiple emulsions contain both types simultaneously. Microemulsions are clear, stable mixtures with particle sizes less than 120nm. The document also discusses emulsifying agents, formulation components, stability issues like flocculation and creaming, and identification tests.
This document defines and describes emulsions. An emulsion is an unstable mixture of two immiscible liquids stabilized by an emulsifying agent. Emulsions can be classified as simple (macro), multiple, or micro. Simple emulsions are oil-in-water or water-in-oil, while multiple emulsions contain both types simultaneously. Microemulsions are clear, thermodynamically stable mixtures containing oil, water, surfactant and sometimes cosurfactant. Emulsions require emulsifying agents, viscosity modifiers, preservatives and sometimes antioxidants for stability. Common emulsifying agents include surfactants, hydrocolloids, and finely divided solids. Instability can occur via flocc
This document summarizes various techniques for staining carbohydrates in tissue sections. It discusses staining methods for glycogen including PAS, PAS with diastase digestion, Best Carmine, and hexamine silver. It also covers staining for acid mucopolysaccharides and glycoproteins using Alcian Blue at pH 2.5 and 1.0, with and without hyaluronidase digestion. Mayer's Mucicarmine stains carboxylated and sulfated mucins. The medical importance of demonstrating glycogen is also mentioned.
The document describes the key steps in the histopathology laboratory workflow. Specimens are received and labeled with patient information. In grossing, samples are selected and inspected. Tissue processing involves dehydration, clearing, impregnation and embedding to prepare samples for microtomy, where thin sections are cut. Sections are stained, usually with H&E, and examined microscopically. Preparation methods include fresh cells/tissues, smears and sectional techniques, with sectional being the standard method used in histopathology.
This document provides an overview of histopathology, including definitions of key terms and descriptions of different tissue sampling methods. It defines histopathology as the study of diseased tissues and cytology as the study of cells from tissues or secretions. The document outlines that a tissue is a group of similar cells that work together, an organ contains multiple tissues, and a system is a group of organs. It also describes different types of biopsies including excisional, incisional, core needle, and endoscopic biopsies. Autopsies are defined as samples taken from deceased individuals to determine cause of death.
This document discusses various biochemical hazard signs that are labeled on bottles of laboratory reagents and chemicals. It lists 9 types of hazard signs: 1) flammable solvents or gas, 2) highly toxic chemicals or gas, 3) oxidizers, 4) corrosive materials, 5) biohazard, 6) radioactive material, 7) laser light, 8) restricted area, and 9) cancer suspect agent. It then provides brief descriptions and examples of substances that would require labels of harmful, flammable, oxidizing, toxic, corrosive, irritating, explosive, and dangerous for the environment. The document concludes by listing additional prohibited acts in laboratories.
This document discusses the importance of laboratory safety and provides guidelines for ensuring personal safety, room safety, and reagent safety. It outlines proper personal protective equipment including lab coats, gloves, and masks. It also describes necessary room features like hazard doors, ventilation, and emergency equipment. The document explains how to safely handle and store reagents, including using material safety data sheets to understand chemical hazards and proper labeling. Methods for analyzing potential hazards are presented. The goal is to educate on creating a safe work environment through protective measures and procedures.
1. Tissue repair involves regeneration of injured tissue or replacement by connective tissue scarring. It involves cell proliferation and interaction between cells and the extracellular matrix.
2. Tissues are divided into continuously dividing, stable, and permanent groups based on their ability to proliferate. Continuously dividing tissues like skin regenerate easily while permanent tissues like neurons cannot regenerate after injury.
3. Growth factors and the extracellular matrix play important roles in tissue repair by stimulating cell growth and movement. Repair occurs through regeneration in labile tissues and scarring in others when injury is too severe for regeneration.
This document discusses frozen sections and cryostats. Frozen sections are prepared without dehydration or embedding to enable rapid diagnosis within 10 minutes. They have applications in intraoperative diagnosis, enzyme histochemistry, immunohistochemistry, and other techniques. Tissue is frozen using liquid nitrogen or other cryogenic methods, turning water within the tissue to ice which acts as an embedding medium for sectioning. Cryostats maintain low temperatures, typically -20 to -30°C, for sectioning frozen tissue blocks. Optimal cutting temperatures vary by tissue type and whether the tissue is fixed.
This document discusses the process of transmission electron microscopy (TEM) specimen preparation. It involves fixing small tissue samples in glutaraldehyde and osmium tetroxide, dehydrating in ethanol, infiltrating with epoxy resin, embedding, sectioning with an ultramicrotome, collecting sections on grids, and staining with heavy metals like uranium and lead. Key steps are fixation at low temperature and pH to best preserve ultrastructure, dehydration and resin infiltration for hardness, and staining for contrast to visualize ultrastructural components by TEM.
This document discusses tissue microarrays (TMAs), which allow analysis of hundreds of tissue samples on a single slide. It describes how TMAs are constructed by taking small tissue cores from donor blocks and embedding them in a recipient block. The advantages of TMAs include high throughput analysis and relatively low cost. Various types of TMAs are used for applications like immunohistochemistry, in situ hybridization, and analyzing protein/DNA expression. The document outlines the steps to construct a TMA, including defining the research question, selecting cases, determining core size and number, making a map, and embedding the cores. Quality controls and limitations are also discussed.
This document provides information on museum techniques for preparing specimens for permanent display and preservation. It discusses the basic steps of reception, preparation, fixation, restoration, preservation, and presentation of specimens. Key techniques include formalin fixation, color restoration methods like Kaiserling and hydrosulphite, and mounting specimens in fluid-filled jars or boxes. Specialized methods are described for tissues, cysts, and friable specimens. The goal is to collect and process specimens in a way that maintains their educational value over the long term.
Chronic inflammation is a prolonged inflammatory response characterized by three key features: greater tissue destruction than acute inflammation, mononuclear infiltrates including lymphocytes and macrophages, and granulation tissue formation and scarring. Chronic inflammation can be caused by persistent infections like tuberculosis or prolonged exposure to toxic agents like silica. Macroscopically, chronic inflammation presents as chronic ulcers, abscesses, thickening of hollow organs, granulomatous lesions, and fibrosis. A granuloma is a distinctive lesion of chronic inflammation containing activated macrophages that fuse to form multinucleated giant cells.
Acute inflammation is the early response of tissues to injury and involves vascular and cellular events. The vascular events include vasodilation, increased vascular permeability allowing plasma proteins to leave circulation, and accumulation of leukocytes from the blood vessels into tissues. The principal leukocytes in acute inflammation are neutrophils. The cellular events in acute inflammation help destroy, dilute or isolate injurious agents. Mediators of acute inflammation include histamine, prostaglandins, nitric oxide, complement factors and cytokines. Acute inflammation is rapid in onset, relatively short in duration and aims to return tissues to normal function.
Pathology is the study of disease through examination of organs, tissues, fluids, and whole bodies. It involves studying the cause, development, structural changes, and clinical significance of disease. When cells are injured, they may adapt, undergo reversible injury, or irreversible injury and death through necrosis or apoptosis. Necrosis results in cell contents spilling out while apoptosis is programmed cell death. At the cellular level, injury can be caused by lack of oxygen, toxins, radiation, and more. Cells attempt to adapt through changes in size, number, or type to cope with stressors.
Lipids are a group of fat-like substances that are insoluble in water but soluble in organic compounds. They include true fats, lipids, sterols, and hydrocarbons. Lipids are classified based on their solubility into simple lipids like fats and oils, compound lipids containing other groups, and derived lipids formed by hydrolysis. Their physical state determines how they behave in staining. Hydrophilic lipids are water-miscible while hydrophobic lipids are not. Special fixation and sectioning are required to demonstrate lipids histochemically. Stains like Oil Red O and Sudan dyes are used to identify lipids in tissues and smears. The distribution and accumulation of lipids are diagnostically important in several body systems and
This document discusses different types of microtomes used to cut extremely thin sections of tissue samples for histology and pathology applications. It describes five main types: rocking microtome, rotary microtome, base sledge microtome, sliding microtome, and freezing microtome. For each type, it provides details on their mechanism of action, advantages, disadvantages and uses. The document also covers microtome parts, knife types and sharpening, factors for good paraffin section cutting, and use of section adhesives.
This document provides information on connective tissues and muscle tissues. It discusses the components and types of connective tissue, including collagen, elastic, and reticular fibers. It also describes the cells found in connective tissue. Special staining techniques for demonstrating these tissues are outlined, including Masson's trichrome, Gomori's trichrome, van Gieson's, and Verhoeff's elastic stains. The document also briefly describes the three types of muscle tissue - skeletal, cardiac, and smooth muscle.
This document discusses proteins and nucleic acids in tissues and various methods for demonstrating them. It covers:
1. Protein composition and types including structural, enzymes, and complement proteins.
2. Methods for demonstrating proteins including immunohistochemistry, histochemical staining of amino acids or enzymes, and physical properties.
3. Nucleic acid composition, structure of DNA and RNA, and their functions in storage of genetic information and protein coding.
4. Techniques for demonstrating DNA including Feulgen reaction, fluorescent staining, and in situ hybridization. Methods for RNA include methyl green-pyronin staining and enzymatic digestion.
This document provides an overview of carbohydrates, including their classification, structures, functions, and histological staining properties. Carbohydrates are classified as simple carbohydrates or glycoconjugates. Glycogen and mucin are two important carbohydrates for histological analysis. Glycogen stains with PAS, Best's carmine, and other techniques. Mucins include acid and neutral forms that stain differently with Alcian blue, PAS, and other histochemical stains depending on their composition. Carbohydrates play important roles in cellular metabolism and structure.
Connective tissue is one of the four main tissue types found throughout the body. It is the most abundant tissue and develops from mesoderm during embryonic development. Connective tissue consists of cells embedded within an extracellular matrix of fibers and ground substance. The major fibers are collagen, elastic, and reticular fibers. Connective tissue provides structure, strength, and binding properties to organs and varies depending on the composition and organization of its cellular and extracellular components.
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বাংলাদেশের অর্থনৈতিক সমীক্ষা ২০২৪ [Bangladesh Economic Review 2024 Bangla.pdf] কম্পিউটার , ট্যাব ও স্মার্ট ফোন ভার্সন সহ সম্পূর্ণ বাংলা ই-বুক বা pdf বই " সুচিপত্র ...বুকমার্ক মেনু 🔖 ও হাইপার লিংক মেনু 📝👆 যুক্ত ..
আমাদের সবার জন্য খুব খুব গুরুত্বপূর্ণ একটি বই ..বিসিএস, ব্যাংক, ইউনিভার্সিটি ভর্তি ও যে কোন প্রতিযোগিতা মূলক পরীক্ষার জন্য এর খুব ইম্পরট্যান্ট একটি বিষয় ...তাছাড়া বাংলাদেশের সাম্প্রতিক যে কোন ডাটা বা তথ্য এই বইতে পাবেন ...
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it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
2. HISTORICAL ASPECT OF
HEMATOXYLIN
•The introduction of hematoxylin is
attributed to Waldeyer in 1862 that
used it as a watery extract but without
very much success.
3. •Two years later Bohmer combined
haematoxylin with alum as a mordant
and obtained more specific staining.
4. •Ehrlich (1886) who overcame the
instability of hematoxylin and alum by
the additions of glacial acetic acid and at
the same time produced his formula for
haematoxylin as it is used today.
5. HAEMATOXYLINS AND
EOSIN (H&E)
•H&E stain is the most popular stain
(routine) in histopathology field.
•Compare the simplicity and demonstrate
clearly different types of tissue
structures.
6. • Hematoxylin stains the nucleus blue-black
with clear chromatin particles.
• Eosin stains cytoplasm and most connective
tissue fibers and muscles in different shades
of colours varying from pink to red.
7. •Hematoxylin is a natural dye extracted
from the log wood (heart wood) of
Haematoxylon campechianum tree.
•Hematoxylin extracted from log wood by
hot water and precipitated by urea.
8. • Hematoxylin original country is southern
Mexico and cultivated for commercial
purposes in Jamaica and Indies.
• Hematoxylin its self is not a stain, unless it
oxidizes to haematein
13. OXIDATION OF
HEMATOXYLIN
•Hematoxylin Oxidized to haematin by
two ways:
Naturally :
•This is a slow process, sometimes taking
as long as 3–4 months, but the resultant
solution seems to retain its staining
ability for a long time.
16. •Chemical oxidation takes short time but
the hematoxylin useful life is short when
compared to naturally oxidized
hematoxylin.
17. •Haematein is an anionic dye having a
poor affinity for tissue ( nucleus), unless
mordant is used.
18. •Mordant with hematoxylins is alkaline
substance (metal), added to haematein to
link between tissue( nuclei) and the dye (
haematein).
19. •Most mordants are incorporated into the
hematoxylin staining solutions, although
certain hematoxylin stains required the
tissue section to be pre-treated with the
mordant before staining; such as
Heidenhain’s iron hematoxylin.
20. TYPES OF HEMATOXYLINS
Hematoxylin solutions can be classified
according to which mordant is used into:
•1. Alum hematoxylins
•2. Iron hematoxylins
22. 1- ALUM HEMATOXYLINS
•Mordant is aluminum either in the form
of aluminum potassium sulfate or
aluminum ammonium sulfate.
•Oxidation either naturally or chemically.
•Stain the cell nucleus red.
23. •Converted into familiar blue color by
process of bluing.
•Bluing is the conversion of the red color
into blue as a result of changing the pH
from acid to alkaline, done by:
24. •R.T.W, 0.05% Ammonia in water,
Alkaline solution such as Lithium
Carbonate, Scott's tap water
25. •The alum hematoxylins can be used
regressively, meaning that the section is
over-stained and then differentiated in
acid alcohol, followed by ‘blueing’,
26. •Or progressively, i.e. stained for a
predetermined time to stain the nuclei
adequately but leave the background
tissue relatively unstained.
27. •The most common alum hematoxylins
are:
•Ehrlich’s H, Delafield H, Mayer’s H,
Harris's H , Cole’s H, Carazzi’s H, and
Gill’s H .
29. •glycerin (stabilizer: prolonged half life
and slow oxidation rate), D.W (solvent),
potassium alum (mordant), and G.A.A
(accentuator)
30. •Uses: good nuclear stain, mucins,
cartilage and bone.
•Advantages: useful for staining sections
from tissues that have been exposed to
acid.
31. •It is suitable for tissues that have been
subjected to acid decalcification or, more
valuably, tissues that have been stored
for along period in formalin fixatives.
46. DISADVANTAGES OF
ALUM H.
•Sensitivity to any subsequently applied
acidic staining solutions .
•This problem overcame by use iron H , or
combination of alum H with Celestine
blue B
47. STAINING TIME WITH ALUM H.
Depend on:
•Type of H used.
•Age of stain.
•Intensity of use of stain.
•Whether the stain used progressively or
regressively
49. EOSIN
•Eosin is the most suitable stain to
combine with an alum hematoxylin to
demonstrate the general histological
architecture of a tissue.
50. •Its particular value is its ability, with
proper differentiation, to distinguish
between the cytoplasm of different types
of cell,
51. • and between the different types of
connective tissue fibers and matrices, by
staining them differing shades of red and
pink.
52. TYPES OF EOSIN
•Eosin B.
•Eosin Y.
•Ethyl eosin.
•The Eosin Y is the most common stain
used as counter stain with hematoxylin,
because it colors back ground by color
vary from pinkish to reddish
53. •1 g or 0.5 g Eosin in 100 ml D.W(1% or
0.5% Aqueous Eosin), 0.05 ml G.A.A and
small amount crystal
thymol(preservative).
54. •Differentiation of the eosin staining
occurs in the subsequent tap water wash,
and a little further differentiation occurs
during the dehydration through the
alcohols.
55. •The intensity of eosin staining, and the
degree of differentiation required, is
largely a matter of individual taste.
56.
57.
58.
59.
60. CELESTINE BLUE-ALUM
HEMATOXYLIN
•Is popular method used overcome
disadvantage of alum hematoxylin
Celestine blue is resistant to the effects of
acid, and the ferric salt in the prepared
Celestine blue solution strengthens
61. •The bond between the nucleus and the
alum hematoxylin to provide a strong
nuclear stain which is reasonably
resistant to acid.
62. IRON HEMATOXYLIN
•In these hematoxylin solutions, iron salts
are used both as the oxidizing agent and
as mordant. The most commonly used
iron salts are ferric chloride and ferric
ammonium sulfate, and the most
common iron hematoxylins are:
63.
64. • Over-oxidation of the hematoxylin is a
problem with these stains, so it is usual to
prepare separate mordant/oxidant and
hematoxylin solutions and mix them
immediately before use e.g. in Weigert’s
hematoxylin)
65. •or to use them consecutively (e.g.
Heidenhain’s and Loyez hematoxylins).
Because of the strong oxidizing ability of
the solution containing iron salts,.
66. •it is often used as a subsequent
differentiating fluid after hematoxylin
staining, as well as for a mordanting
fluid before it
67. •The iron hematoxylins are capable of
demonstrating a much wider range of
tissue structures than the alum
hematoxylins, but the techniques are
more time-consuming.
69. WEIGERT,S HEMATOXYLIN
• This is an iron hematoxylin in which ferric
chloride is used as the mordant/oxidant. The
iron and the hematoxylin solutions are
prepared separately and are mixed
immediately before use. Used to stain nuclei
70. Preparation:
The iron and hematoxylin solutions are
prepared separately and are mixed
immediately before use.
Solution A:
1 g hematoxylin dissolve in 100 ml of absolute
alcohol
71. •Solution B (Mordant and oxidizing):
•30% ferric chloride 4ml
•Conc Hcl 1ml
•D.W 95 ml
72. The color of the mixture should be a violet
black. If muddy – brown, it must be
discarded.
Differentiator used 1% acid alcohol
73. HEIDENHAIN’S HEMATOXYLIN
•This iron hematoxylin uses ferric
ammonium sulfate as oxidant/mordant,
and the same solution is used as the
differentiating fluid.
74. The iron solution is used first
The section is treated with hematoxylin
solution until it is over stained,
Then it is then differentiated with iron
solution under microscopic control.
76. • It may be used to demonstrate
chromatin,
chromosomes,
nuclei,
centrosomes,
mitochondria,
muscle striations
myelin
77. LOYEZ HEMATOXYLIN
• This iron hematoxylin uses ferric ammonium
sulfate as the mordant. The mordant and
hematoxylin solutions are used consecutively,
and differentiation is by Weigert’s
differentiator ((borax and potassium
ferricyanide)
78. •It is used to demonstrate myelin and can
be applied to paraffin, frozen, or
nitrocellulose sections.
79. VERHÖEFF’S HEMATOXYLIN
•This iron hematoxylin is used to
demonstrate elastic fibers. Ferric chloride
is included in the hematoxylin staining
solution,
80. •together with Lugol’s iodine, and 2%
aqueous ferric chloride is used as the
differentiator. Coarse elastic fibers stain
black.
81. TUNGSTEN HEMATOXYLINS
• Mallory phosphotungstic acid hematoxylin
(PTAH) is only one widely used tungsten
hematoxylin. combined hematoxylin with 1%
aqueous phosphotungstic acid, the latter
acting as the mordant.
82. •Its use is applicable to both CNS material
and general tissue structure, and to
tissues fixed in any of the standard
fixatives.
84. LEAD HEMATOXYLINS
• Hematoxylin solutions that incorporate lead
salts have recently been used in the
demonstration of the granules in the
endocrine cells of the alimentary tract and
other regions.
85. • The most practical diagnostic application is in
the identification of endocrine cells in some
tumors, but it is also used in research
procedures such as in the localization of
gastrin-secreting cells in stomach.
86. HEMATOXYLIN WITHOUT A
MORDANT
•Freshly prepared hematoxylin solutions,
used without a mordant, have been used
to demonstrate various minerals in tissue
sections (Iron, Copper).
87. •The basis of the method is the ability of
hematoxylin to form blue black lakes
with these metals
88. TEST FOR STAINING POWER OF
HEMATOXYLIN
•Adding few drops of hematoxylin to
50ml of tap water will turn a bright,
clear purple or blue violet color.
•Exhausted solutions will not be clear &
bright & the color will be rusty or green
89.
90. APPLICATION OF H&E
•1-Cell biology
•2-Primary diagnostic technique in the
histopathology laboratory.
•3-Primary technique for the evaluation
of morphology.
93. TROUBLESHOOTING OF H& E
STAIN
Problem Causes Solvents
White spots are seen
in the section after
deparaffinization
step. If they are not
recognized at this
point, spotty or
irregular staining
will be seen
microscopically on
the stained section.
A. The section was
not dried properly
before beginning
deparaffinization.
B. the slide did not
remain in xylene
long enough for
complete removal of
the paraffin.
A. The slides must
be treated with
absolute alcohol to
remove the water
and then retreated
with xylene to
remove the paraffin.
If incomplete drying
is severe, the
sections may loosen
from the slides.
94. B. The slides should be
returned to xylene for a
longer
time.
The nuclei are too pale (the
hematoxylin is too light).
A. The sections were not
stained long enough in
hematoxylin.
B. The hematoxylin was
over oxidized and should
not have been used.
C. The differentiation step
was too long.
D. Pale nuclei in bone
sections may be the result
of over decalcification
A. The section must be
restrained. When sections
have been placed in an
extremely acidic fixative
such as Zenker solution,
the ability to stain the
nucleus may be
impaired and the time in
the hematoxylin may have
to be increased, or a
method to increase tissue
basophilia may be needed.
95. B. Discard hematoxylin
and replace with fresh.
C. Run back and restrain
D. no solution
The nuclei are overstained
(the hematoxylin is too
dark), or
diffuse hematoxylin
staining of the cytoplasm
has occurred
A. The sections were
stained too long in
hematoxylin.
B. The sections are too
thick.
C. The differentiation step
was too short.
A. Decolorize the section
and restain, making
appropriate adjustments in
the staining time of
hematoxylin.
B. Recut the section.
C. . Decolorize the section
and restain, making
appropriate adjustments in
the differentiation times
96. Red or red-brown nuclei. A. The hematoxylin is
breaking down.
B. The sections were not
blued sufficiently
A. Check the oxidation
status of the hematoxylin.
B. Allow a longer time for
bluing of the sections; it is
impossible to over blue the
sections.
Pale staining with eosin. A. The pH of the eosin
solution may be above 5.0,
possibly caused by
carryover of the bluing
reagent.
B. The sections may be too
thin.
C. Slides may have been
left too long in the
dehydrating
solutions
A. Check the pH of the
eosin solution, and adjust it
to a pH of 4.6 to 5.0 with
acetic acid if necessary. Be
sure the bluing reagent is
completely removed before
transferring the slides to
the eosin.
B. Check the thickness of
the section.
97. C. Restain with eosin and
do not allow the stained
slides to stand in the lower
concentrations of alcohols
Cytoplasm is overstained,
and the differentiation is
poor.
A. The eosin solution may
be too concentrated,
especially if
phloxine is present.
B. The section may have
been stained for too long.
C. The sections may have
been passed through the
dehydrating alcohols too
rapidly for good
differentiation of the
eosin to occur.
A. Dilute the eosin
solution.
B. Decrease the staining
time.
C. Allow more time in each
of the dehydrating
solutions for
adequate differentiation of
the eosin.
(Also, check the section
thickness.)
98. Blue—black precipitate on
top of the sections.
The metallic sheen that
develops on most
hematoxylin
solutions has been picked
up on the slide
Filter the hematoxylin
solution daily before
staining
slides
Water bubbles are seen
microscopically in the
stained
sections.
The sections were not
completely dehydrated,
and
water is present in the
mounted section.
Remove the cover glass
and mounting medium
with xylene. Return the
slide to fresh absolute
alcohol (several changes).
After the sections are
dehydrated, clear with
fresh xylene and mount
with synthetic resin.
All dehydrating and
clearing solutions should
be changed before staining
any more sections
99. Difficulty bringing some
areas of the tissue in focus
with
light microscopy.
Mounting medium may be
present on top of the cover
glass.
Remove the cover glass
and remount with a clean
cover glass. Review the
method used for mounting
sections, and modify if
needed
The mounting medium has
retracted from the edge of
the
cover glass.
A. The cover glass is
warped.
B. The mounting medium
has been thinned too much
with xylene.
A. Remove the cover glass
and apply a new cover
glass.
B. Apply a new cover glass
with fresh mounting
medium. Keep the
mounting medium
container tightly
capped when not in use.
Use a small container for
the mounting medium and
discard when it becomes
too thick
100. The water and the slides turn
milky when the slides are
placed in the water following
the rehydrating alcohols.
Xylene has not been removed
completely by the
alcohols
Change the alcohols, back the
slides up to absolute
alcohol, and rehydrate the
sections
The slides are hazy or milky
in the last xylene rinse prior
to coverslipping.
Water has not been
completely removed from the
sections before being placed
in the xylene.
Change the alcohol solutions,
especially the anhydrous
or absolute reagents.
Redehydrate the sections and
clear in fresh xylene
The mounted stained sections
do not show the usual
transparency and crispness
when viewed by light
microscopy.
The mounting medium may
be too thick, causing the
cover glass to be held too far
above the tissue.
Remove the cover glass and
mounting medium with
xylene. Remount the section
with fresh mounting
medium.