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.
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.
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 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 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
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
Special stains are used in hematology and cytology to identify specific molecules that are not visible with routine staining. They can determine if certain molecules are present or absent, where they are located, and how much is present. Examples of special stains discussed in the document include fetal hemoglobin stain, Prussian blue for sideroblasts, methyl violet for Heinz bodies, Papanicolaou stain in cytology, and Masson Fontana silver stain to identify melanin and argentaffin granules. These stains help characterize and diagnose blood and cellular abnormalities.
This document discusses special stains used in histopathology, specifically stains used to identify lipids. It describes the Oil Red O and Sudan Black B stains. Oil Red O stains neutral triglycerides and lipids red and nuclei blue. It is used to identify tumors containing fat cells and fatty emboli. Sudan Black B stains fats blue-black and nuclei red. It can identify phospholipids and is used to differentiate myeloblasts from lymphoblasts in hematological disorders. Special stains are important tools to identify tissue components not visible with routine H&E staining.
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.
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.
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 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 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
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
Special stains are used in hematology and cytology to identify specific molecules that are not visible with routine staining. They can determine if certain molecules are present or absent, where they are located, and how much is present. Examples of special stains discussed in the document include fetal hemoglobin stain, Prussian blue for sideroblasts, methyl violet for Heinz bodies, Papanicolaou stain in cytology, and Masson Fontana silver stain to identify melanin and argentaffin granules. These stains help characterize and diagnose blood and cellular abnormalities.
This document discusses special stains used in histopathology, specifically stains used to identify lipids. It describes the Oil Red O and Sudan Black B stains. Oil Red O stains neutral triglycerides and lipids red and nuclei blue. It is used to identify tumors containing fat cells and fatty emboli. Sudan Black B stains fats blue-black and nuclei red. It can identify phospholipids and is used to differentiate myeloblasts from lymphoblasts in hematological disorders. Special stains are important tools to identify tissue components not visible with routine H&E staining.
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.
Gomori Reticular Fiber Stain RESEARCH 04-2016 PowerpointLawrence Reynolds
This document describes research testing different reducing agents in Gomori's reticulin silver stain. Gomori's stain is used to visualize reticulin fibers in tissues like liver, which helps diagnose diseases. The study tested various reducing agents like formaldehyde, glutaraldehyde, and acids to see which provided the best contrast of reticulin fibers. Results found that non-buffered formaldehyde and glutaraldehyde produced staining similar to the standard, while buffered solutions and weak acids showed less fiber contrast. The conclusions were that the best reducing agents can slowly oxidize to lower pH and reduce silver deposits on fibers for optimal visualization.
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
The PAS stain demonstrates carbohydrates and carbohydrate-rich compounds in tissues through oxidizing glycol groups with periodic acid and forming a magenta-colored complex with Schiff's reagent. It is useful for detecting conditions like glycogen storage disease and assessing thickness of the glomerular basement membrane. The PAS stain demonstrates substances like mucins, fungi cell walls, gangliosides, lipofuscin, Russell bodies and the basement membranes of various tissues.
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 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,
Romanowsky stains are commonly used to stain blood films and identify blood components. Some key Romanowsky stains discussed in the document include Leishman, Giemsa, Wright's, Field, Jenner, and JSB stains. These stains involve using dyes like methylene blue and eosin in specific combinations and concentrations to differentially stain structures in blood films based on their chemical properties. Proper staining technique and protocols are outlined to clearly identify red blood cells, white blood cells, parasites, and other components when examining stained blood films under a microscope.
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.
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.
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 various carbohydrate stains including periodic acid Schiff (PAS), Alcian blue, mucicarmine, and others. It describes the principles, procedures, and results of PAS staining for glycogen and fungi. Different types of mucins are discussed along with combined staining techniques to differentiate neutral from acidic mucins. The document also covers connective tissue stains for collagen and elastic fibers, as well as special stains for pigments, minerals, and lipids.
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 provides information on staining blood films and smears. It discusses the different types of stains used including Romanowsky stains like Leishman stain, Giemsa stain, Wright stain, and Field stain. Specimens should be collected in EDTA and smears prepared within an hour then fixed in methanol or ethanol to preserve cell morphology before staining. Romanowsky stains use methylene blue and eosin dyes to reveal subtle differences in cell structures and components.
Tissue Processing for Histopathological AnalysisKomal Parmar
This document provides an overview of histological and histopathological tissue processing techniques. It discusses the key steps in processing tissue, including fixation, dehydration, clearing, embedding, sectioning, and staining. Proper tissue processing is important for microscopic analysis and involves techniques to harden tissues without distortion, remove water, and infiltrate paraffin wax to allow thin sectioning. Automated tissue processors can standardize and expedite the multi-step procedure.
This document discusses enzymes and enzyme histochemistry techniques. It defines enzymes and their classifications including hydrolases, oxidoreductases, transferases, and lyases. It then demonstrates the histochemical localization of alkaline phosphatase and acid phosphatase in tissues. The optimal fixation, incubation, and interpretation of results are described. Finally, it lists some diagnostic applications of enzyme histochemistry and provides sample multiple choice questions.
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.
The document summarizes the process of tissue processing which involves fixing, dehydrating, clearing, infiltrating with wax, embedding, sectioning, staining, and mounting tissue samples in order to examine them microscopically. Key steps include fixation to prevent decay, dehydration using alcohol to remove water, clearing with xylene to remove alcohol, infiltration and embedding in paraffin wax, sectioning thin slices with a microtome, staining typically with hematoxylin and eosin for examination, and mounting on slides. The goal is to prepare tissue for microscopic analysis while maintaining structure.
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.
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.
Gomori Reticular Fiber Stain RESEARCH 04-2016 PowerpointLawrence Reynolds
This document describes research testing different reducing agents in Gomori's reticulin silver stain. Gomori's stain is used to visualize reticulin fibers in tissues like liver, which helps diagnose diseases. The study tested various reducing agents like formaldehyde, glutaraldehyde, and acids to see which provided the best contrast of reticulin fibers. Results found that non-buffered formaldehyde and glutaraldehyde produced staining similar to the standard, while buffered solutions and weak acids showed less fiber contrast. The conclusions were that the best reducing agents can slowly oxidize to lower pH and reduce silver deposits on fibers for optimal visualization.
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
The PAS stain demonstrates carbohydrates and carbohydrate-rich compounds in tissues through oxidizing glycol groups with periodic acid and forming a magenta-colored complex with Schiff's reagent. It is useful for detecting conditions like glycogen storage disease and assessing thickness of the glomerular basement membrane. The PAS stain demonstrates substances like mucins, fungi cell walls, gangliosides, lipofuscin, Russell bodies and the basement membranes of various tissues.
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 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,
Romanowsky stains are commonly used to stain blood films and identify blood components. Some key Romanowsky stains discussed in the document include Leishman, Giemsa, Wright's, Field, Jenner, and JSB stains. These stains involve using dyes like methylene blue and eosin in specific combinations and concentrations to differentially stain structures in blood films based on their chemical properties. Proper staining technique and protocols are outlined to clearly identify red blood cells, white blood cells, parasites, and other components when examining stained blood films under a microscope.
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.
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.
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 various carbohydrate stains including periodic acid Schiff (PAS), Alcian blue, mucicarmine, and others. It describes the principles, procedures, and results of PAS staining for glycogen and fungi. Different types of mucins are discussed along with combined staining techniques to differentiate neutral from acidic mucins. The document also covers connective tissue stains for collagen and elastic fibers, as well as special stains for pigments, minerals, and lipids.
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 provides information on staining blood films and smears. It discusses the different types of stains used including Romanowsky stains like Leishman stain, Giemsa stain, Wright stain, and Field stain. Specimens should be collected in EDTA and smears prepared within an hour then fixed in methanol or ethanol to preserve cell morphology before staining. Romanowsky stains use methylene blue and eosin dyes to reveal subtle differences in cell structures and components.
Tissue Processing for Histopathological AnalysisKomal Parmar
This document provides an overview of histological and histopathological tissue processing techniques. It discusses the key steps in processing tissue, including fixation, dehydration, clearing, embedding, sectioning, and staining. Proper tissue processing is important for microscopic analysis and involves techniques to harden tissues without distortion, remove water, and infiltrate paraffin wax to allow thin sectioning. Automated tissue processors can standardize and expedite the multi-step procedure.
This document discusses enzymes and enzyme histochemistry techniques. It defines enzymes and their classifications including hydrolases, oxidoreductases, transferases, and lyases. It then demonstrates the histochemical localization of alkaline phosphatase and acid phosphatase in tissues. The optimal fixation, incubation, and interpretation of results are described. Finally, it lists some diagnostic applications of enzyme histochemistry and provides sample multiple choice questions.
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.
The document summarizes the process of tissue processing which involves fixing, dehydrating, clearing, infiltrating with wax, embedding, sectioning, staining, and mounting tissue samples in order to examine them microscopically. Key steps include fixation to prevent decay, dehydration using alcohol to remove water, clearing with xylene to remove alcohol, infiltration and embedding in paraffin wax, sectioning thin slices with a microtome, staining typically with hematoxylin and eosin for examination, and mounting on slides. The goal is to prepare tissue for microscopic analysis while maintaining structure.
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.
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.
H and E staining is most important part of the histopathological diagnosis, this presentation is to highlight some important basic concept of the Staining.
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.
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.
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.
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.
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.
This document discusses principles of staining tissues for microscopic examination. It focuses on hematoxylin and eosin (H&E) staining, which is the most widely used staining method. Hematoxylin is a basic dye that stains nuclei blue-black, while eosin is an acidic dye that stains cytoplasm and other structures pink. For hematoxylin to effectively stain nuclei, it must be oxidized to hematein and combined with a mordant like aluminum to increase its affinity for tissue. H&E staining allows clear differentiation of structures and is simple to perform while providing diagnostic information.
Glossary of staining methods, reagents, immunostaining, terminology and eponymsPravin Amabade
This document provides a glossary of terms related to biological staining methods, reagents, immunostaining, and related terminology. It contains over 150 entries defined in black bold, with cross-references in underlined blue italic. The glossary aims to define terms for researchers across sub-disciplines of microtechnique to increase understanding of staining rationales, reagents, and eponyms. It is updated periodically to expand coverage of dyes, fixatives, and other procedures.
Notes for STAINING AND ANALYSIS of HISTOLOGICAL PREPARATIONSimprovemed
This document provides an overview of histological staining techniques. It discusses how histological preparations are stained using interactions between dyes, solvents, and tissue components. Different staining methods result in different colors that highlight various structures. A classic example is hematoxylin and eosin staining, where hematoxylin stains acidic components blue and eosin stains basic components pink. Specialized staining techniques also exist, such as immunohistochemistry. Proper staining selection depends on the tissue and research goals. Histological preparations are then analyzed under a microscope to study cell and tissue morphology.
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 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.
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 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.
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.
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.
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
Cellular adaptation involves alterations in cell structure and function in response to sublethal stimuli. There are several types of morphologic adaptation:
Hypertrophy is an increase in cell size without cell division. Hyperplasia is an increase in cell number. Atrophy is a decrease in cell size and number. Metaplasia is the replacement of one differentiated cell type with another. Dysplasia involves abnormal cell growth and maturation. These adaptive responses allow cells to withstand sublethal stimuli but may progress to more severe injury if the stimuli persist.
This document provides an overview of histopathology techniques, including different types of biopsy methods, principles of specimen handling and examination, tissue processing steps, and staining procedures. It discusses incisional, excisional, punch, core needle, and curettage biopsies. Key steps in tissue processing are described as fixation, dehydration, clearing, embedding, sectioning, and staining. Common stains like H&E and special stains are also outlined.
Osteogenic cells are rounded cells with rounded nuclei that are rich in alkaline phosphatase and present in the endosteum. They cannot divide and have an acidophilic cytoplasm. Osteogenic cells are found in the endosteum. Osteoclasts are large, multinucleated cells that are found in Howship's lacunae and have foamy acidophilic cytoplasm containing lysosomes. They are derived from monocytes. Haversian systems in compact bone are interconnected by interstitial lamellae and contain concentric lamellae, central Haversian canals, and osteocytes in lacunae.
Osteogenic cells are rounded cells with rounded nuclei that are rich in alkaline phosphatase. They are present in the endosteum and are acidophilic cells. Osteogenic cells cannot divide. Osteoclasts are large, multinucleated cells that are found in Howship's lacunae and have foamy acidophilic cytoplasm containing lysosomes. They are derived from monocytes. Haversian systems in compact bone are interconnected by interstitial lamellae and contain concentric lamellae, osteocytes in lacunae, and a central Haversian canal.
This document discusses the musculoskeletal system and provides details on cartilage, bone, and ossification. It describes three types of cartilage - hyaline, yellow elastic fibrocartilage, and white fibrocartilage. It also outlines the structure and function of compact and cancellous bone, as well as the cells and tissues involved in bone formation like osteoblasts, osteocytes, osteoclasts, periosteum, and endosteum. Finally, it summarizes the two types of ossification - intramembranous and endochondral/intracartilagenous ossification.
This document discusses the musculoskeletal system and provides details on cartilage, bone, and ossification. It describes the three types of cartilage - hyaline, elastic, and fibrocartilage - and their structure and locations in the body. It also outlines the structure of compact and spongous bone, including the cells and layers involved. Finally, it explains the two types of ossification - intramembranous and endochondral - and the stages of each process.
This document summarizes the structure and function of the musculoskeletal system. It describes the three types of cartilage - hyaline, elastic, and fibrocartilage. It also discusses the structure of bones, including compact and spongy bone. Finally, it examines the two processes of bone formation: intramembranous ossification which forms flat bones, and endochondral ossification where cartilage is replaced by bone in long bones.
The document discusses statistical analysis and statistical software packages like SPSS. It explains concepts like hypothesis, level of significance, statistical tests like ANOVA and t-tests. It provides examples of hypothesis statements, how to conduct one-way and two-way ANOVA, and the steps involved in statistical analysis and research proposals. Factor analysis is introduced as a statistical method to describe variability among observed correlated variables in terms of fewer unobserved factors.
The document provides guidance on preparing the Results section of a research paper. It recommends that the Results section:
- Summarize the key findings without providing excessive detail
- Present results objectively without interpretation
- Highlight important findings in text and use tables and figures to complement rather than repeat the data
- Use the past tense and cite results clearly while referring to tables and figures
Detection systems are used in immunohistochemistry and in-situ hybridization to detect bound primary antibodies or nucleic acid probes, using enzymes, substrates, and chromogens to produce a visible signal under microscopy; common detection systems are based on horseradish peroxidase or alkaline phosphatase enzymes with DAB or permanent red chromogens, while in-situ hybridization localizes DNA or RNA in tissue using labeled probes. Blocking steps help reduce background staining when using these detection systems.
The document provides guidance on writing the results section of a research paper. The key points are:
1) The results section should summarize the main findings of the study in relation to the research objectives, without providing extensive details or interpreting the results.
2) Visuals like tables and figures are generally better than text for presenting data findings. The text should highlight important points and complement the visuals without repeating all the information.
3) The results section only reports what was found and presents data objectively, leaving interpretation and discussion of what the results mean for the discussion section. It answers the question "what happened" based on analysis of the collected data.
This document provides guidance on writing the methodology and results sections of a research report. It discusses that the methodology section must provide enough detail that others could replicate the study, and should include subsections on participants, materials, and procedure. The participants subsection describes the sample. The materials subsection describes any tools or stimuli used. The procedure subsection outlines the steps of the experiment. The results section should report key findings through descriptive statistics, tables, and graphs. Statistical analyses and their results should also be presented.
This document summarizes the histology of bone. It describes bone as a mineralized connective tissue that provides structure and protection. The key components of bone are osteoblasts, osteocytes, osteoclasts, and a matrix containing collagen and calcium. Osteoblasts form new bone, osteocytes are embedded in the matrix, and osteoclasts resorb bone. Bone is further organized into compact bone, with dense osteons, and cancellous or spongy bone, with thin trabeculae. Bone remodeling is mediated by these cells to maintain bone strength and mineral homeostasis.
This document discusses the histology of cartilage. It defines cartilage and its main functions, which include bearing mechanical stresses, forming frameworks, and facilitating bone movement. The document then describes the three main types of cartilage - hyaline, elastic, and fibrocartilage - and discusses their composition, distribution in the body, and characteristics. It also addresses cartilage growth and repair mechanisms as well as common conditions that affect cartilage.
This document provides information about analyzing cerebrospinal fluid (CSF). It discusses the formation and function of CSF and describes routine laboratory assays performed on CSF samples, including examinations of appearance, cell counts, chemical analysis, and microbiology. Procedures for collecting and handling CSF samples are outlined. Normal ranges and pathological conditions associated with abnormal CSF results are also reviewed.
Microorganisms can be classified based on their size, shape, and cellular structure. Bacteria are single-celled organisms that can be further classified as cocci, bacilli, or spirochetes depending on their shape. Special stains like Gram stain and acid-fast stain are used to differentiate bacteria and identify medically important types. Fungi have cell walls containing chitin while viruses are protein-coated genes that need host cells. Protozoa are single-celled organisms that move using pseudopods, flagella, or cilia. A variety of staining techniques exist to identify bacteria, fungi, and other microorganisms in clinical samples and tissue sections.
This document discusses various biological, infectious, chemical, mechanical, and ergonomic hazards found in laboratory environments. It outlines policies and procedures for implementing safety precautions like universal precautions, personal protective equipment, exposure control plans, hazard communication standards, and proper handling of sharps, waste and hazardous chemicals. Specific guidance is provided for pathogens such as HIV, hepatitis viruses, tuberculosis, and prion diseases. Engineering and administrative controls are emphasized to reduce risks from all identified hazards.
This document appears to be a series of 10 case studies presenting cytology samples from fine needle aspirations and other specimens. Each case includes images of cell morphologies and characteristics along with multiple choice options for possible diagnoses. In case 7, images show poorly cohesive epithelial-like cells associated with fibromyxoid stroma, suggesting a diagnosis of adenoid cystic carcinoma. Case 10 features images consistent with Warthin's tumor, characterized by lymphocytes and oncocytic cells. The document seems to be a teaching aid to help learners identify cell types and arrive at cytological diagnoses.
This document provides guidance on writing effective multiple choice questions (MCQs) for assessment. It discusses the benefits of MCQs, such as wide topic coverage and fast feedback, as well as disadvantages like guessing and lower-order thinking. Tips are provided for writing high-quality question stems and plausible distractors that avoid clues. The document emphasizes shifting focus from recall to application and using novel contexts to make questions less "googleable." Strategies are presented for engaging students with formative assessment through varied feedback, social learning, and emphasis on intrinsic motivation.
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2. The Hematoxylin and Eosin stain (H&E) is the most widely used
histological stain because :
comparative simplicity
Ability to demonstrate clearly an enormous number of different tissue
structures.
Hematoxylin stains cell nuclei blue black shows good intranuclear detail.
Eosin stains cell cytoplasm and most connective tissue fibers in varying
shades and intensities of pink, orange, and red.
3. Stains chemical substances used to achieve
visible color contrast in the microscopic
picture of a prepared tissue
Staining treating tissues or cells with a
series of reagents so that it acquires a color;
no particles of dye are seen and the stained
element is transparent.
4. Outlines tissues and cellular components.
Identification of tissues.
Establishes the presence or absence of
disease processes.
5. Most commonly used staining methods are –
Hematoxylin and Eosin staining in Histopathology
Gram’s Stain and Ziehl-Neelson staining in Microbiology.
Romanowsky staining in Hematology.
Papanicoloau staining in Cytology
6. Essentially Aromatic benzene ring
compounds or derivatives that possess the
twin properties of color and ability to bind
to tissue.
7. Categories based on Examples
Origins Natural (Hematoxylin & Carmine)
Synthetic
Aniline
Physiochemical Properties Fluorescent (acridine orange)
Leuco (Leuco methylene blue)
Metachromatic (toluidiene blue)
Neutral (azure-eosinate)
Structure Azo (orange)
Metal complex (Al or Fe complexes of
hematein)
Xanthene (pyronine Y)
Use in biological staining Fat (oil red O)
Flourescent probe(YOYO-1)
Mucin (alcian blue)
Use in textile dyeing Acid (eosin)
Basic (safrarine)
Direct (Congo Red)
Supposed mode of action
of dye
Mordant (Gallacyarine chrome alum
Reactive(mercury orange)
8. Acidic Dyes
Stains basic components such as
cytoplasm, acidiophil granules, etc
Eosin, Acid Fuchsin
Basic Dyes
Stains acidic components such as
nucleus, basophil granules,etc.
Hematoxylin, Basic Fuchsin, Methylene
Blue.
Neutral Dyes
Consists of mixtures of basic and
acidic dyes.
Both cations and anions contain
chromophoric groups and both have
colored radicals.
Romanowsky dyes formed by
interaction of polychrome methylene
blue and eosin
9. 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.
Should not be used to indicate any substance that improves
in staining in some other manner (accentulators and
accelerators)
10. The mordant dye complex ‘lake’ combines
with tissue to form tissue-mordant-dye
complex, which is insoluble.
11. 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.
12. Accentuators:
Substances which increase the staining power of dye.
They increase the intensity & selectivity of stain.
e.g KOH in Lofflers methylene blue
phenol in carbol fuschin & carbol thionin.
Accelerators
Accentuators used in metallic impregnation technique for the
nervous system.
e.g chloral hydrate
Trapping agents
Agents which holds dye combination with tissue or bacteria .
e.g tannic acid/iodine
13. 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.
15. Vital Staining Applied to living tissue
Accomplished by injecting staining solution into
some part of the body
Mixing of stain with living cells
Primarily used for research.
Routine Staining Stains tissues with minimal differentiation
except between nucleus and cytoplasm.
Demonstrates general relationship among
cells, tissues and organs.
e.g Hematoxylin and Eosin stains
Special Staining Demonstrates selective features of tissues :
Particular cell products.
Microscopic intracellular and intercellular
structure.
e.g PAS stain for mucopolysaccharide.
16. Regressive Staining
•Tissue is initially overstained and then partially decolorized
(differentiated) until the proper endpoint is reached.
• Sharper degree of differentiation is obtained
•The differentiation is controlled visually by microscopic
examination.
•Faster and more convenient .
Progressive Staining
•Tissue is stained for a predetermined time for adequate
staining of the nuclei and leaves the background tissue relatively
unstained.
•Once the dye is taken up by the tissues, it is not removed.
•The tissue is left in the dye solution until it retains the desired
amount of coloration.
•The differentiation solely relies on the selective affinity of dyes
for different tissue elements.
17. Removal or washing out of
excess stain until the color
is retained only by the tissue
component that is to be
studied.
Done with acid alcohol or
ethyl alcohol
Exposure to air may oxidize
and improve the process.
18. All glassware should be thoroughly cleaned.
Correct solvent should be used.
Silver and osmic acid solutions should be kept in dark bottles.
Solutions like dilute ammonia should be freshly prepared.
Constituents of stain dissolved should follow the formula.
Alcoholic solutions of the stain should be kept in dark stoppered
bottles.
All dyes should be filtered before use.
19. Specially designated bench
Staining bench should be facing the
window.
Slide washing tray made of stainless
steel.
Bunsen burner to heat up the stain.
Thermostatically controlled hot place to
melt the wax.
Microscope to control staining reaction.
20. Slides are stained using –
Using staining dishes
Small grooved coplin jars with lids.
Large staining troughs
Using staining racks
2 pieces of stout rods 2-4 cm apart.
Using staining machine
Same as processing machine but
carry slide racks.
21. The word Hematoxylin is derived
from old Greek word Haimato
meaning blood & Xylon meaning
wood.
A natural dye extracted from the
core or heartwood of tree
Haematoxylon campechianum.
The hematoxylin is extracted from
logwood with hot water, and then
precipitated out from the aqueous
solution using urea.
The major oxidization product of
hematoxylin is hematein, a natural
dye that is responsible for the
color properties.
22. Waldeyer firmly established the use of
hematoxylin in histology in 1862.
Bohmer combined hematoxylin with
alum as a mordant and obtained more
specific staining in 1864.
Heidenhan introduced his classical Iron-
Alum- Hematoxylin method used as a
standard technique in cytology.
Ehrlich overcame the instability of
hematoxylin and alum by adding glacial
acteic acid and simultaneously
produced his formula for hematoxylin
used today.
23. The process of oxidation of hematoxylin.
The major oxidization product is hematein, a natural dye that is
responsible for the color properties.
It is a poor dye but metallic mordant and forms the most powerful
stain.
Carried out in 2 ways –
Natural oxidation.
Chemical oxidation.
24. Natural Oxidation
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.
Advantages Disadvantage
Once oxidation has reached an
acceptable level, the staining
solution may be used and it
lasts for longer.
•Requires a considerable period
of time.
•Two batches of naturally
ripened product may not
produce the same staining
qualities.
e.g Ehrlich’s & Delafield’s hematoxylin solutions
25. Achieved by addition of oxidizing
agents such as mercuric oxide,
sodium iodate and potassium
permanganate.
This process converts the
hematoxylin haematin almost
instantaneously, so these
hematoxylin solutions are ready for
use after preparation.
26. Properties of chemically oxidized hematoxylin
Shorter useful life than the naturally oxidized haematoxylins.
The process of over-oxidation of hematoxylin has established
that the production of oxyhaematein inhibits successful
staining.
Glycerol has been incorporated in many formulas as it’s a
stabiliser to prevent over-oxidation and prevent evaporation.
27. Properties of chemically oxidized hematoxylin
Haematein is anionic, having a poor affinity for tissue, and is inadequate as a
nuclear stain without the presence of a mordant.
The mordant/metal cation confers a net positive charge to the dye-mordant
complex and enables it to bind to anionic tissue sites, such as nuclear
chromatin.
The type of mordant used influences strongly the type of tissue components
stained and their final color.
The most useful mordants for hematoxylin are salts of aluminum, iron, and
tungsten, although hematoxylin solutions using lead as a mordant are
occasionally used in the demonstration of argyrophil cells).
28. Hematoxylin solutions can be arbitrarily classified
according to which mordant is used:
Alum hematoxylins
Iron hematoxylins
Tungsten hematoxylins
Molybdenum hematoxylins
Lead hematoxylins
Hematoxylin without mordant.
29. Used routinely in the hematoxylin and eosin stain
and produce good nuclear staining.
The mordant is aluminum in the form of ‘potash
alum - aluminum potassium sulfate or ‘ammonium
alum -aluminum ammonium sulfate
Alum Hematoxylin can be used progressively or
regressively.
31. Naturally ripened strong alum hematoxylin.
Stains nuclei intensely and crisply - stained sections fade much more
slowly .
Stains mucin in salivary glands, cartilage and cement lines of bones
Suitable for tissues subjected to acid decalcification.
Suitable for tissues that have been stored for a long period in formalin
fixatives which have gradually become acidic over the storage period.
Suitable for Bouin’s fixed tissue.
not ideal for frozen sections.
32. Preparation :
Hematoxylin 2 g
Absolute alcohol 100 ml
Glycerol 100 ml
Distilled water 100 ml
Glacial acetic acid 10 ml
Potassium alum 15 g approx.
33. Dissolve the hematoxylin in alcohol
Incorporate glycerol to slow down the oxidation process and prolong
shelf time.
Add potassium alum till saturation.
The stain may be ripened naturally by allowing to stand in a large
flask, loosely stoppered with cotton wool – takes about 2 months.
In case of emergency, the stain could be chemically ripened by the
addition of sodium iodate using 50mg for every gram of
hematoxylin.
Filter before use.
34. A widely used hematoxylin stain.
Chemically ripened with sodium iodate.
More vigorous in action than Ehrlich’s hematoxylin.
Used as both progressive and regressive stain.
Used as a nuclear counterstain in the demonstration of glycogen
(PAS,mucicarmine) in various enzyme histological techniques.
Stain applied for a short period - 5-10 mins until nuclei are stained and
then blued without any differentiation which might destroy/decolor the
stained cytoplasmic components.
35. Preparation
Hematoxylin 1 g
Distilled water 1000 ml
Potassium or ammonium alum 50 g
Sodium iodate 0.2 g
Citric acid 1 g
Chloral hydrate SLR 50 g
or
Chloral hydrate AR 30 g
36. The hematoxylin, potassium alum, and sodium iodate are
dissolved in distilled water by warming and stirring, or by
allowing to stand at room temperature overnight.
Chloral hydrate and citric acid are added & the mixture is
boiled for 5 minutes, then cooled and filtered.
Chloral hydrate acts as a preservative and citric acid sharpens
nuclear staining
37. This alum hematoxylin was traditionally chemically ripened with mercuric
oxide (sodium or potassium iodate is frequently used as a substitute for
oxidation).
It gives particular clear nuclear staining.
It is used as a regressive stain in routine histology practice.
It is used as a progressive stain in diagnostic exfoliative cytology.
When using it as a progressive stain, an acetic acid-alcohol rinse provides a
more controllable method in removing excess stain from tissue components
and the glass slide.
38. Preparation
Hematoxylin 2.5 g
Absolute alcohol 25 ml
Potassium alum 50 g
Distilled water 500 ml
Mercuric oxide 1.25 g
or
Sodium iodate 0.5 g
Glacial acetic acid 20 ml
39. Dissolve hematoxylin in alcohol
Add it to alum previously dissolved in warm distilled water
The mixture is rapidly brought to boil
Mercuric oxide is then slowly and carefully added, when the solution turns dark purple.
The stain is rapidly cooled under tap water.
Optional addition of glacial acetic acid.
Filter before use.
40. Available in 3 concentrations –
◦ Gill’s I (normal)
◦ Gill’s II (double)
◦ Gill’s III (triple) most concentrated.
More frequently used than Mayer’s hematoxylin for routine H&E
staining.
More stable than Harris’s hematoxylin, as auto-oxidation is inhibited
to the extent.
41. Preparation of solution
Hematoxylin 2 g
Sodium iodate 0.2 g
Aluminum sulfate 17.6 g
Distilled water 750 ml
Ethylene glycol (ethandiol) 250 ml
Glacial acetic acid 20 ml
42. Advantages
Fast in action.
Stable for at least months.
Produce little or no surface
precipitate.
Their preparation doesn’t
involve boiling the solution.
Disadvantages
Staining of gelatin is
adhesive and even the glass
itself.
Some mucus may also stain
darkly, as compared to
Harris’s hematoxylin.
43. Alum hematoxylin, artificially ripened with an alcoholic iodine
solution.
Has good keeping qualities and is suitable for use especially in
sequence with celestine blue unlike Ehrlich’s hematoxylin
45. The hematoxylin is dissolved in warm distilled water and mixed
with iodine solution.
The alum solution is added and the mixture brought to boil
then cooled quickly and filtered.
The solution is ready for immediate use, but may need on
occasion filtering after storage.
46. Alum hematoxylin which is chemically ripened using
potassium iodate
Used as a progressive nuclear counterstain.
Largely confined to use with frozen sections because it gives
excellent and clear nuclear staining with a very short staining
time.
47. Preparation –
Hematoxylin 5 g
Glycerol 100 ml
Potassium alum 25 g
Distilled water 400 ml
Potassium iodate 0.1 g
48. Hematoxylin is dissolved in the glycerol
Alum is dissolved in most of the water overnight.
The alum solution is added slowly to the hematoxylin solution.
Potassium iodate is dissolved in the rest of the water with gentle
warming & is then added to the haematoxylin-alum-glycerol
mixture.
The final staining solution is mixed well and is then ready for
immediate use;
It remains usable for about 6 months.
49. A naturally ripened alum hematoxylin with similar longevity to
Ehrlich’s hematoxylin.
Preparation
Hematoxylin 4 g
95% alcohol 125 ml
Saturated aqueous ammonium alum 400 ml
(15 g/100 ml)
Glycerin 100 ml
50. The hematoxylin is dissolved in 25 ml of alcohol,
and then added to the alum solution.
This mixture is allowed to stand in light and air for 5
days and then filtered.
Glycerin and a further 100 ml of 95% alcohol are
added to this mixture.
Allow the stain to stand exposed to light and air for
about 3–4 months or until sufficiently dark in color.
Filter before use.
51. Staining time varies according to various factors:
Type of hematoxylin used
e.g. Ehrlich’s hematoxylin - 20–45 minutes
Mayer’s hematoxylin - 10–20 minutes.
Age of stain.
As the stain ages, the staining time will need to be increased.
Intensity of use of stain.
A heavily used hematoxylin will lose its staining powers more
rapidly and longer staining times will be necessary.
Whether the stain is used progressively or regressively
e.g. Mayer’s hematoxylin used progressively 5–10 minutes and
used regressively 10–20 minutes.
52. Pre-treatment of tissues or sections.
e.g. length of time in fixative or acid decalcifying solution or
whether paraffin or frozen sections.
Post-treatment of sections
e.g. subsequent acid stains such as van Gieson.
Personal preference.
53. 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 prepared 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.
55. 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 before use
57. In these hematoxylins, iron salts such as ferric chloride and ferric
ammonium sulfate are used both as the oxidizing agent and as
mordant.
Over-oxidation of the hematoxylin is a problem with these
stains.
Mordant/oxidant and hematoxylin solution are prepared
separately and mixed before use.
Capable of demonstrating wider range of tissue structures
compared to alum haematoxylin.
Techniques are more time consuming and needs microscopic
control for accuracy.
58. An iron hematoxylin used as a nuclear stain in techniques
where acidic staining solutions are applied to the sections
subsequently
e.g Van Gieson stain – picric acid is a constituent which have
marked decolorizing action on nuclei stained with alum
hematoxylin.
It is a useful stain, with eosin, for CNS tissues.
59. Preparation
a) Hematoxylin solution
Hematoxylin 1 g
Absolute alcohol 100 ml
This is allowed to ripen naturally for 4 weeks before use.
b) Iron solution
30% aqueous ferric chloride (anhydrous) 4 ml
Hydrochloric acid (concentrated) 1 ml
Distilled water 95 ml
60. This iron hematoxylin uses ferric ammonium sulfate as oxidant/mordant
It is used as the differentiating fluid.
It is a cytological stain.
It is used regressively.
After staining, all components are black or dark gray-black.
The hematoxylin staining is removed progressively from different tissue
structures at different rates using the iron alum solution.
It may be used to demonstrate
Chromatin
Chromosomes
Nuclei
Centrosomes
Mitochondria
Muscle striations
Myelin
61. Preparation
a. Hematoxylin solution
Hematoxylin 0.5 g
Absolute alcohol 10 ml
Distilled water 90 ml
The hematoxylin is dissolved in the alcohol, and the water is then added.
The solution is allowed to ripen naturally for 4 weeks before use.
b. Iron solution (5% iron alum)
Ferric ammonium sulfate 5 g
Distilled water 100 ml
It is important that only the clear violet crystals of ferric ammonium sulfate be used.
62. This iron hematoxylin uses ferric ammonium sulfate as the
mordant.
Differentiation is by Weigert’s differentiator (borax and
potassium ferricyanide).
Used to demonstrate myelin.
Can be applied to paraffin, frozen, or nitrocellulose sections.
63. This iron hematoxylin is used to demonstrate elastic fibers after all
routine fixative.
Ferric chloride is included in the hematoxylin staining solution,
together with Lugol’s iodine, and 2% aqueous ferric chloride is used
as the differentiator.
Coarse elastic fibres stain black, but the staining of fine fibers may
be less than satisfactory.
The differentiation step is critical to the success of this method.
64. Widely used tungsten hematoxylin is PTAH
(Phosphotungstic acid hematoxylin
Technique).
Used to demonstrate fibrin, muscle striations,
cilia and glial fibres.
Myelin can also be demonstrated
Widely used as a CNS stain.
65. Preparation
PTAH solution using haematin
Haematin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
Stain is ready to use immediately, but short-lived.
PTAH Solution (KMnO4)
Haematoxylin 0.59 g
Phosphotungstic acid 5g
Distilled water 500ml
0.25% Aqueous KMnO4 25 ml
Peak staining activity after 7 days
66. Results
Muscle striations / neuroglia fibres / fibrin / amoeba – Dark Blue.
Nuclei/cilia/RBC – Blue
Myelin – Lighter blue
Collagen /Osteoid / Cartilage / Elastic fibres – Deep brownish red.
Cytoplasm – Pale pinkish brown.
67. Hematoxylin solution using molybdic acid as
mordant.
Rare stain
Used in demonstration of collagen, coarse reticulin.
Also stains Argentaffin cell granules.
68. Preparation
a. Hematoxylin solution
Hematoxylin 2.5 g
Dioxane 49 ml
Hydrogen peroxide 1 ml
b. Phosphomolybdic acid solution
Phosphomolybdic acid 16.5 g
Distilled water 44 ml
Diethylene glycol 11 ml
The resultant dark violet solution is allowed to stand for 24 hours
before use.
69. Results –
Collagen and coarse reticulin - violet to black
Argentaffin cells - black
Nuclei pale - blue
Paneth cells - orange
Tissue fixed in dichromate do not give good results.
70. Used in demonstration of granules in
endocrine cells of ailmentary tract and other
regions.
Most practical diagnostic application is in
identification of endocrine cells in tumors of
doubtful origin.
Also used in localisation of gastrin secreting
cells in stomach.
71. Freshly prepared hematoxylin is used to
demonstrate various minerals in tissue
sections.
These methods are now replaced with more
specific techniques.
72. Xanthine dyes which stains connective tissue and cytoplasm in
varying intensity and shades (red to pink).
Available in the following types :
Eosin Y ( Eosin Yellowish, Eosin water soluble) – most widely available.
Ethyl Eosin (Eosin S, eosin alcohol soluble).
Eosin B ( Eosin Bluish, Erythrosine B).
Ethyl eosin and eosin B are now rarely used, although occasional old
methods specify their use – e.g the Harris stain for Negri bodies.
73. Eosin Y
Most commonly used eosin.
Readily soluble in water.
Satisfactorily soluble in alcohol.
Preparation
Eosin Y, water soluble 5 gm
Distilled water 1000 ml
Crystals of Thymol added to inhibit fungal growth.
Addition of little acetic acid (0.5 -1000 ml stain) sharpens the
staining.
74. Principle
Hematoxylin and Eosin are principle stains used for
demonstration of nucleus and cytoplasm.
Alum acts as a mordant and the hematoxylin containing alum
stains the nucleus light blue which turns red in the presence
of acid.
The cell differentiation is achieved by treating the tissue with
acid solution.
The counterstaining is performed using eosin which imparts
pink color to cytoplasm.
75. 1. REMOVAL OF WAX.
2. HYDRATION WITH GRADED ALCOHOLS.
3. STAINING.
4. DIFFERENTIATION
5. BLUEING
6. COUNTERSTAIN WITH EOSIN
7. DEHYDRATION THROUGH GRADED ALCOHOL.
8. CLEARING IN XYLENE
9. MOUNTING UNDER A COVER SLIP.
76. REMOVAL OF WAX
Removal of wax with xylene (impermeable to stains).
2-3 minutes of xylene immersion sufficient for sections of
10µ thickness.
Facilitated by warming the slides at 60 degrees oven to melt
the wax.
77. HYDRATION WITH GRADED ALCOHOLS
Sections are transferred to absolute alcohol for 1-2 minutes –
til it becomes opaque.
Sections rinsed in 2nd bath of alcohol, drained and taken to
water.
Any pigments or deposits should be removed at this stage.
78. STAINING
Slides immersed in hematoxylin (Mayers / Harris/ Gills)
If regressive stain is used, longer time is required to overstain
the structures
79. DIFFERENTIATION
Sections are dipped in acid alcohol, agitated and washed in
tap water.
Observed under microscope
If underdifferentiated – returned to acid alcohol.
If overdifferentiated – returned to hematoxylin and
differentiation repeated.
80. BLUEING
Slides after draining off hematoxylin is transferred to ammonia water for 2
minutes.
Section when removed from hematoxylin or acid alcohol are pink in color.
Washing turns them blue Blueing.
Blueing solutions are usually preferred alkaline .
a)Ammonium hydroxide in 70% alcohol
b) lithium carbonate stock solution
c) Scott’s tap water
Magnesium sulfate (MgSO4) 30.0 gm
Sodium bicarbonate 2.0 gm
Tap water 3000.0 ml
d) tap water pH - 7
COUNTERSTAIN WITH EOSIN
Transfer the slides to 1% aqueous eosin for 2 minutes.
Wash in running water.
81. DEHYDRATION THROUGH GRADED ALCOHOL.
After staining the sections are transferred to 90% alcohol and
agitated for 10 seconds followed by
then to absolute alcohol 1for (10-15 sec) followed by
absolute alcohol 2 for 30 sec.
82. Slides are transferred to xylene 1 and left until completely clear . It
should betestedfor clarity.
Then its transferred to xylene 2which they be mounted.
83. MOUNTING
Its required to maintain high refractive index necessary for
microscopy and to protect the sections during storage.
METHOD :
A drop the mountant is placed on the section , place the
convenient sized coverslip into position.
(Air bubbles may be removed by gentle pressure on the coverslip)
MOUNTANTS :
Aqueous mountants
Resinous mountants
84. USES :
• To be used with metachromatic dyes.
• Standard mountant for fat tissues.
Low refracting index of 1.4 – 1.42.
Glycerin is added to prevent cracking and splitting of
dyes.
Bacteriostatic agent should be added (thymol).
Types :
• Gelatin media
• Gum arabic media
Apathy’s medium – fluorescent microscopy
Highman’s modified apathy’s medium
Farrant’s medium
Fructose syrup
85. Its composed of natural or synthetic resins.
Stained preparations are most transparent when the
refractive index is 1.54.
Natural resins :
◦ Canada balsam dissolved in xylol to 55-70%.
◦ Dammar balsam.
◦ Colophonium resin – used in alcoholic solutions.
◦ Terpene resin.
Synthetic resins :
◦ Plasticizers such as tricreysl phosphate or dibutyl
phatalate is added.
◦ Commonly used is kirkpatrick and lendrum’s DPX
87. To coat the edges of coverslip , so that no air bubbles develop.
Commonly used are :
◦ Paraffin wax
◦ Cement
◦ Varnish
88. Results
Nuclei appear blue/black in color
Cytoplasm appears in varying shades of pink
Muscle fibers appear deep pink/red in color
Red blood cells appear orange/red in color
Fibrin appears deep pink in color.
89. 1. HYDRATION WITH GRADED ALCOHOLS.
2. STAINING.
3. DIFFERENTIATION
4. BLUEING
5. COUNTERSTAIN WITH EOSIN
6. DEHYDRATION THROUGH GRADED ALCOHOL.
7. CLEARING IN XYLENE
8. MOUNTING UNDER A COVER SLIP.
90. 1. Remove polyethylene glycol fixative in 50% alcohol, 2 minutes.
2. Hydrate in 95% alcohol, 2 minutes, and 70% alcohol, minutes.
3. Rinse in water, 1 minute.
4. Stain in Harris’s hematoxylin, 5 minutes.
5. Rinse in water, 2 minutes.
6. Differentiate in 0.5% aqueous hydrochloric acid,10 seconds ,approx.
7. Rinse in water, 2 minutes.
8. ‘Blue’ in Scott’s tap water substitute, 2 minutes.
91. 9. Rinse in water, 2 minutes.
10. Dehydrate, 70% alcohol for 2 minutes.
11. Dehydrate, 95% alcohol, 2 minutes.
12. Dehydrate, 95% alcohol, 2 minutes.
13. Stain in OG 6, 2 minutes.
14. Rinse in 95% alcohol, 2 minutes.
15. Rinse in 95% alcohol, 2 minutes.
16. Stain in EA 50, 3 minutes.
17. Rinse in 95% alcohol, 1 minute.
92. Results
Nuclei appear blue/black in color.
Cytoplasm (non-keratinizing squamous cells) appear
blue/green in color.
Keratinizing cells appear pink/orange in color.
93. 1. Freeze suitable tissue block onto a chuck.
2. Cut cryostat sections at 3–6 μm thickness.
3. Fix section in 10% neutral buffered formalin at room temperature for 20
seconds.
4. Rinse in tap water.
5. Stain in double strength Carazzi’s hematoxylin for 1 minute.
6. Wash well in tap water for 10–20 seconds.
7. Stain in 1% aqueous eosin for 10 seconds.
8. Rinse in tap water.
9. Dehydrate, clear, and mount.
94. 1.
2.
3.
4.
5.
6.
7.
8.
9.
Dewax and hydrate paraffin sections, removing mercury precipitate if indicated.
Oxidize for 5 minutes in 0.5% aqueous periodic acid.
Rinse in tap and then in distilled water.
Place in Schiff’s reagent for 15 minutes ( 10 minutes for frozen sections).
Rinse for 2 minutes in each of three changes of freshly made sulfite rinse.
Wash 5 to 10 minutes in running tap water.
Optional counterstain with Harris’ hematoxylin for 1 -3 minutes or in light green (0.1% in
0.1% acetic acid) for 5-20 seconds. Light green is especially useful when searching for
fungi.
If hematoxylin is used, differentiate by means of 3-5 quick dips in 1% acid alcohol, wash in
tap water and blue in Scott’s tap water substitute; then wash 5 minutes in running water.
Dehydrate, clear and mount.