This document discusses cytopreparatory techniques for cytology samples. It describes the different types of cytology samples like exfoliative cytology, aspiration cytology, and body fluids. The key steps in cytopreparatory techniques are outlined as evaluation of specimens, preparation of smears, fixation, and staining. Factors that can affect optimal cytological preparation like quality of specimen, fixative used, and stains are also summarized. Different fixation techniques including dry, wet, liquid-based, and lysing fixation for bloody samples are explained.
Gross Examination, Selection, Collection and Fixation of Specimen ghulam abbas
The document discusses the gross examination, selection, collection and fixation of specimens in pathology. It covers identifying and labeling specimens, performing a gross examination, selecting relevant portions for microscopic examination, and principles of proper fixation. Fixation preserves tissue morphology and prevents autolysis and contamination. Common fixatives include 10% neutral buffered formalin, Bouin's solution, B5, and Zenker's solution. Proper handling and fixation are important for accurate laboratory diagnosis.
Quality Control In Histopathology Dr.Rami amawi.pptxRami Al Amawi
This document discusses quality control in histopathology. It defines quality assurance, quality control, and quality improvement. It outlines the pre-analytic, analytic, and post-analytic phases of quality control and common problems and solutions in each phase. It also discusses approaches to quality control like intradepartmental consultation, random case review, clinical indicators, pathology turnaround times, and monitoring specimen adequacy.
This document outlines the steps for histology lab procedures which include fixation, processing, sectioning, staining, and mounting of tissues. It lists the required chemicals, glassware, and instruments. The key steps are:
1. Tissues are fixed in formalin or Bouin's fixative for 24 hours to preserve cells and structures.
2. Processing involves dehydration using graded alcohols, clearing with benzene, and infiltration with paraffin wax for embedding.
3. Sections are cut from the wax blocks using a microtome, mounted on slides, and stained with H&E stain.
4. Stained sections are viewed under a microscope after dewaxing and mounting.
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.
Automation in histopathology or advance technology in histopathology labSamim Bashir
now a day all work is done by machines .there is alot of new machines which work autmatically in hisopathology.they reduce human effort .they also increase the speed of work flow
processing of bone marrow trephine biopsykanwalpreet15
there is no standard method for processing of bone marrow trephine biopsies. there are various fixatives and decalcifying agents . depending upon need of IHC and cytogenetics, we can decide
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
This document discusses cytopreparatory techniques for cytology samples. It describes the different types of cytology samples like exfoliative cytology, aspiration cytology, and body fluids. The key steps in cytopreparatory techniques are outlined as evaluation of specimens, preparation of smears, fixation, and staining. Factors that can affect optimal cytological preparation like quality of specimen, fixative used, and stains are also summarized. Different fixation techniques including dry, wet, liquid-based, and lysing fixation for bloody samples are explained.
Gross Examination, Selection, Collection and Fixation of Specimen ghulam abbas
The document discusses the gross examination, selection, collection and fixation of specimens in pathology. It covers identifying and labeling specimens, performing a gross examination, selecting relevant portions for microscopic examination, and principles of proper fixation. Fixation preserves tissue morphology and prevents autolysis and contamination. Common fixatives include 10% neutral buffered formalin, Bouin's solution, B5, and Zenker's solution. Proper handling and fixation are important for accurate laboratory diagnosis.
Quality Control In Histopathology Dr.Rami amawi.pptxRami Al Amawi
This document discusses quality control in histopathology. It defines quality assurance, quality control, and quality improvement. It outlines the pre-analytic, analytic, and post-analytic phases of quality control and common problems and solutions in each phase. It also discusses approaches to quality control like intradepartmental consultation, random case review, clinical indicators, pathology turnaround times, and monitoring specimen adequacy.
This document outlines the steps for histology lab procedures which include fixation, processing, sectioning, staining, and mounting of tissues. It lists the required chemicals, glassware, and instruments. The key steps are:
1. Tissues are fixed in formalin or Bouin's fixative for 24 hours to preserve cells and structures.
2. Processing involves dehydration using graded alcohols, clearing with benzene, and infiltration with paraffin wax for embedding.
3. Sections are cut from the wax blocks using a microtome, mounted on slides, and stained with H&E stain.
4. Stained sections are viewed under a microscope after dewaxing and mounting.
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.
Automation in histopathology or advance technology in histopathology labSamim Bashir
now a day all work is done by machines .there is alot of new machines which work autmatically in hisopathology.they reduce human effort .they also increase the speed of work flow
processing of bone marrow trephine biopsykanwalpreet15
there is no standard method for processing of bone marrow trephine biopsies. there are various fixatives and decalcifying agents . depending upon need of IHC and cytogenetics, we can decide
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
The document summarizes a seminar presentation on histopathology staining techniques. It discusses the routine H&E stain and provides details on the principle, reagents, and procedure. It also describes special stains used to identify substances like carbohydrates, amyloid, nucleic acids, and lipids. Specific stains covered include periodic acid Schiff, Congo red, Feulgen, methyl green pyronin, and Oil red O. The document aims to inform attendees about common and special staining methods in histopathology.
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.
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.
The LE cell demonstration document describes the LE cell, which is a neutrophil that has phagocytosed nuclear material coated with antinuclear antibodies, a characteristic of lupus erythematosus. It discusses several methods for demonstrating LE cells in blood samples, including using clotted blood, defibrinated blood, or the rotary method. The rotary method involves adding glass beads to heparinized blood and rotating at 50rpm for 30 minutes at 37 degrees Celsius before preparing buffy coat smears to identify LE cells.
This document provides information on general histopathology techniques. It defines histology and histopathology, and describes the process of collecting biopsy specimens including fixation, storage, and labeling. It discusses different types of biopsies like core needle, fine needle, excisional, and incisional biopsies. Common histological techniques are also outlined, such as frozen section, fine needle aspiration, and exfoliative cytology. Immunohistochemistry is also briefly described.
This document discusses various methods of decalcification used to remove calcium from bones and calcified tissues for histological examination. It describes the main decalcification methods including acid decalcification using strong acids like hydrochloric acid and nitric acid or weak acids like formic acid. Other methods discussed are chelating agents like EDTA, ion-exchange resins, and electrolysis. The document also covers factors affecting decalcification rate and various tests to determine the endpoint of decalcification including radiographic, chemical, and physical examination of the tissue.
Microtomes, Section cutting , Sharpening of Razorsvikas25187
This document discusses various types of microtomes and microtomy techniques. It describes different parts of microtomes like the block holder, knife holder, and handwheels. It explains different types of microtomes based on their cutting mechanism, including rotary, rocking, base-sledge, sliding, freezing, vibrating, saw, cryostat, and ultramicrotome. It also discusses microtome knives, sharpening techniques, section cutting for paraffin blocks, and section mounting methods.
There are several knife profiles used for microtomy including Profile A (plano concave), Profile B (biconcave), Profile C (wedge), and Profile D (tool edge). Profile A has one flat side and one concave side and is used for soft tissues embedded in nitrocellulose. Profile B is classical with concavity on both sides and was introduced by Heiffor. Profile C is a standard wedge profile used for cutting all materials in all microtomes. Profile D is a wedge knife with a steep cutting edge used for hard objects like bone.
Frozen sections of tissue are prepared using a cryostat to quickly obtain thin sections for histological examination and diagnosis. A cryostat maintains tissue at freezing temperatures to allow sectioning without ice crystal formation. Tissue is mounted on a chuck and placed in the cryostat, then sectioned and mounted on slides for staining. Frozen sections allow rapid diagnosis but have poorer morphology and staining than fixed tissue sections.
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 an overview of the process of histopathology. It discusses the key steps: sample collection and fixation, processing including dehydration, clearing, infiltration and embedding, sectioning of samples, and staining. Common techniques and agents used at each step are outlined, such as formalin for fixation, alcohol and acetone for dehydration, xylene for clearing, and paraffin for infiltration and embedding. Hematoxylin and eosin staining is described as the standard staining method, along with Pap staining and Toluidine blue staining. Finally, storage of slides and blocks in refrigerated boxes is mentioned.
This document discusses quality control in histopathology. It defines key terms like quality control, quality assurance, and total quality management. It outlines the pre-analytical, analytical and post-analytical phases of quality control and highlights variables that can affect quality in each phase like personnel training, equipment, and interpretation of results. It provides recommendations for achieving quality control through standardized procedures, monitoring turnaround times, participation in proficiency testing, and review of reports. The goal is to generate accurate histopathology reports and enable easy retrieval if needed.
This document discusses histopathology and the process of tissue fixation. It defines histopathology as the study of diseased tissues to examine changes in structure from disease. The key steps in tissue fixation are described, including the objectives to preserve tissue structure and prevent decomposition. Various types of fixatives are classified and their mechanisms and properties explained, with examples like formalin, glutaraldehyde, alcohol, picric acid and osmium tetraoxide. Compound fixatives are also mentioned.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
The document discusses tissue fixation and fixatives. It defines a fixative as a substance that prevents post-mortem changes and preserves the morphological and chemical characteristics of cells and tissues. The aims of fixation include preserving tissues as close to their living state as possible and preventing autolysis and bacterial attack. Common methods of fixation discussed are immersion, perfusion, heat, and vapor fixation. Types of fixatives covered include formaldehyde, alcohols, picric acid, mercuric chloride, and glutaraldehyde. Factors that influence fixation quality like fixation time and temperature are also addressed.
The document discusses various techniques for preparing cell blocks (CBs) from cytology specimens such as effusions, fine needle aspirations, and scrapings. Traditional methods involved using a celloidin or agar embedding medium but newer automated techniques using filters and cassettes provide higher cellularity. CBs allow morphological examination and ancillary studies to improve diagnostic accuracy compared to smears alone. While useful, CBs require more material and time than smears and may lack sufficient cells for all tests.
The document provides information about Hematoxylin and Eosin staining. It states that H&E staining is widely used because of its simplicity, ability to clearly demonstrate tissue structures, and how it specifically stains cell nuclei blue-black and cytoplasm pink to red. Hematoxylin stains nuclei while Eosin stains cytoplasm. The document then discusses the chemistry and preparation of hematoxylin solutions, including how it is extracted from logwood and oxidized to its active form hematein. It also covers the use of mordants like aluminum, iron, and tungsten to improve hematoxylin's staining ability.
The document discusses the principles and techniques of tissue infiltration and embedding. It describes how clearing agents are removed from tissues through diffusion and replaced with molten embedding media like wax. The wax is then cooled to solidify and provide support for sectioning thin tissue samples. Several factors influence infiltration including tissue size, type, clearing agent used, and whether vacuum embedding is performed. Common embedding media include paraffin wax which provides good support but can cause shrinkage, and DMSO-supplemented wax which speeds infiltration. Both manual and automated processors are described.
This document discusses the equipment used in a histopathology lab. It describes microscopes, microtomes, paraffin wax baths, slide warmers, and knives for sectioning tissues. Tissues are embedded in cassettes of different colors and stored in cabinets. Automatic tissue processors are used for dehydrating and embedding tissues in wax. The overall purpose is to examine tissue samples microscopically to study disease manifestations.
Histological techniques involve preparing tissue for microscopic examination through a series of procedures to retain the tissue's microscopic anatomy and allow for good staining. This involves fixing, processing, embedding, sectioning, and staining the tissue. The key steps are fixation to prevent deterioration, dehydration and clearing to infiltrate the tissue with wax, sectioning thin slices, and staining, most commonly with hematoxylin and eosin, to highlight structures. The goal is to represent the tissue's structure as closely as possible to its in vivo state.
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxAnthonyMatu1
This document provides information on the processes involved in preparing histological specimens, including tissue fixation, processing, sectioning, and staining. It describes the objectives of tissue fixation as preventing autolysis and bacterial attack while maintaining tissue structure. The main types of fixatives and factors affecting fixation are outlined. Tissue processing involves dehydration using graded alcohols, clearing with agents like xylene, and embedding in paraffin wax. Sections are cut with a microtome and stained, with hematoxylin and eosin being a common staining method that colors nuclei blue and cytoplasm pink. Other specialized staining techniques are also mentioned.
The document summarizes a seminar presentation on histopathology staining techniques. It discusses the routine H&E stain and provides details on the principle, reagents, and procedure. It also describes special stains used to identify substances like carbohydrates, amyloid, nucleic acids, and lipids. Specific stains covered include periodic acid Schiff, Congo red, Feulgen, methyl green pyronin, and Oil red O. The document aims to inform attendees about common and special staining methods in histopathology.
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.
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.
The LE cell demonstration document describes the LE cell, which is a neutrophil that has phagocytosed nuclear material coated with antinuclear antibodies, a characteristic of lupus erythematosus. It discusses several methods for demonstrating LE cells in blood samples, including using clotted blood, defibrinated blood, or the rotary method. The rotary method involves adding glass beads to heparinized blood and rotating at 50rpm for 30 minutes at 37 degrees Celsius before preparing buffy coat smears to identify LE cells.
This document provides information on general histopathology techniques. It defines histology and histopathology, and describes the process of collecting biopsy specimens including fixation, storage, and labeling. It discusses different types of biopsies like core needle, fine needle, excisional, and incisional biopsies. Common histological techniques are also outlined, such as frozen section, fine needle aspiration, and exfoliative cytology. Immunohistochemistry is also briefly described.
This document discusses various methods of decalcification used to remove calcium from bones and calcified tissues for histological examination. It describes the main decalcification methods including acid decalcification using strong acids like hydrochloric acid and nitric acid or weak acids like formic acid. Other methods discussed are chelating agents like EDTA, ion-exchange resins, and electrolysis. The document also covers factors affecting decalcification rate and various tests to determine the endpoint of decalcification including radiographic, chemical, and physical examination of the tissue.
Microtomes, Section cutting , Sharpening of Razorsvikas25187
This document discusses various types of microtomes and microtomy techniques. It describes different parts of microtomes like the block holder, knife holder, and handwheels. It explains different types of microtomes based on their cutting mechanism, including rotary, rocking, base-sledge, sliding, freezing, vibrating, saw, cryostat, and ultramicrotome. It also discusses microtome knives, sharpening techniques, section cutting for paraffin blocks, and section mounting methods.
There are several knife profiles used for microtomy including Profile A (plano concave), Profile B (biconcave), Profile C (wedge), and Profile D (tool edge). Profile A has one flat side and one concave side and is used for soft tissues embedded in nitrocellulose. Profile B is classical with concavity on both sides and was introduced by Heiffor. Profile C is a standard wedge profile used for cutting all materials in all microtomes. Profile D is a wedge knife with a steep cutting edge used for hard objects like bone.
Frozen sections of tissue are prepared using a cryostat to quickly obtain thin sections for histological examination and diagnosis. A cryostat maintains tissue at freezing temperatures to allow sectioning without ice crystal formation. Tissue is mounted on a chuck and placed in the cryostat, then sectioned and mounted on slides for staining. Frozen sections allow rapid diagnosis but have poorer morphology and staining than fixed tissue sections.
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 an overview of the process of histopathology. It discusses the key steps: sample collection and fixation, processing including dehydration, clearing, infiltration and embedding, sectioning of samples, and staining. Common techniques and agents used at each step are outlined, such as formalin for fixation, alcohol and acetone for dehydration, xylene for clearing, and paraffin for infiltration and embedding. Hematoxylin and eosin staining is described as the standard staining method, along with Pap staining and Toluidine blue staining. Finally, storage of slides and blocks in refrigerated boxes is mentioned.
This document discusses quality control in histopathology. It defines key terms like quality control, quality assurance, and total quality management. It outlines the pre-analytical, analytical and post-analytical phases of quality control and highlights variables that can affect quality in each phase like personnel training, equipment, and interpretation of results. It provides recommendations for achieving quality control through standardized procedures, monitoring turnaround times, participation in proficiency testing, and review of reports. The goal is to generate accurate histopathology reports and enable easy retrieval if needed.
This document discusses histopathology and the process of tissue fixation. It defines histopathology as the study of diseased tissues to examine changes in structure from disease. The key steps in tissue fixation are described, including the objectives to preserve tissue structure and prevent decomposition. Various types of fixatives are classified and their mechanisms and properties explained, with examples like formalin, glutaraldehyde, alcohol, picric acid and osmium tetraoxide. Compound fixatives are also mentioned.
This is a presentation covering all techniques in histopathology. Comprehensive coverage of all related aspects.. Useful for postgraduate Pathology students and practitioners.
The document discusses tissue fixation and fixatives. It defines a fixative as a substance that prevents post-mortem changes and preserves the morphological and chemical characteristics of cells and tissues. The aims of fixation include preserving tissues as close to their living state as possible and preventing autolysis and bacterial attack. Common methods of fixation discussed are immersion, perfusion, heat, and vapor fixation. Types of fixatives covered include formaldehyde, alcohols, picric acid, mercuric chloride, and glutaraldehyde. Factors that influence fixation quality like fixation time and temperature are also addressed.
The document discusses various techniques for preparing cell blocks (CBs) from cytology specimens such as effusions, fine needle aspirations, and scrapings. Traditional methods involved using a celloidin or agar embedding medium but newer automated techniques using filters and cassettes provide higher cellularity. CBs allow morphological examination and ancillary studies to improve diagnostic accuracy compared to smears alone. While useful, CBs require more material and time than smears and may lack sufficient cells for all tests.
The document provides information about Hematoxylin and Eosin staining. It states that H&E staining is widely used because of its simplicity, ability to clearly demonstrate tissue structures, and how it specifically stains cell nuclei blue-black and cytoplasm pink to red. Hematoxylin stains nuclei while Eosin stains cytoplasm. The document then discusses the chemistry and preparation of hematoxylin solutions, including how it is extracted from logwood and oxidized to its active form hematein. It also covers the use of mordants like aluminum, iron, and tungsten to improve hematoxylin's staining ability.
The document discusses the principles and techniques of tissue infiltration and embedding. It describes how clearing agents are removed from tissues through diffusion and replaced with molten embedding media like wax. The wax is then cooled to solidify and provide support for sectioning thin tissue samples. Several factors influence infiltration including tissue size, type, clearing agent used, and whether vacuum embedding is performed. Common embedding media include paraffin wax which provides good support but can cause shrinkage, and DMSO-supplemented wax which speeds infiltration. Both manual and automated processors are described.
This document discusses the equipment used in a histopathology lab. It describes microscopes, microtomes, paraffin wax baths, slide warmers, and knives for sectioning tissues. Tissues are embedded in cassettes of different colors and stored in cabinets. Automatic tissue processors are used for dehydrating and embedding tissues in wax. The overall purpose is to examine tissue samples microscopically to study disease manifestations.
Histological techniques involve preparing tissue for microscopic examination through a series of procedures to retain the tissue's microscopic anatomy and allow for good staining. This involves fixing, processing, embedding, sectioning, and staining the tissue. The key steps are fixation to prevent deterioration, dehydration and clearing to infiltrate the tissue with wax, sectioning thin slices, and staining, most commonly with hematoxylin and eosin, to highlight structures. The goal is to represent the tissue's structure as closely as possible to its in vivo state.
PREPARATION OF HISTOLOGICAL SPECIMENS.pptxAnthonyMatu1
This document provides information on the processes involved in preparing histological specimens, including tissue fixation, processing, sectioning, and staining. It describes the objectives of tissue fixation as preventing autolysis and bacterial attack while maintaining tissue structure. The main types of fixatives and factors affecting fixation are outlined. Tissue processing involves dehydration using graded alcohols, clearing with agents like xylene, and embedding in paraffin wax. Sections are cut with a microtome and stained, with hematoxylin and eosin being a common staining method that colors nuclei blue and cytoplasm pink. Other specialized staining techniques are also mentioned.
This document summarizes the process of histopathology tissue processing and slide preparation. It discusses manual and automatic tissue processing, embedding tissues in paraffin blocks, microtome sectioning of thin tissue slices, mounting slices on slides with an adhesive, and staining slides using hematoxylin and eosin dyes. The staining procedure involves dewaxing, rehydrating, hematoxylin staining of nuclei, differentiation, bluing, eosin counterstaining, dehydration, clearing, and mounting coverslips for microscopic examination.
This document discusses the process of tissue processing in histology and histopathology laboratories. [1] Tissue samples are obtained from biopsies and autopsies and undergo histotechniques to prepare them for microscopic examination. [2] The key steps include fixation, processing, embedding in paraffin wax, sectioning, staining, and mounting. [3] Automated equipment is now commonly used to improve efficiency at many steps such as tissue processing, sectioning, and staining.
1. The document discusses the various steps involved in tissue processing for microscopic examination, which includes fixation, processing, embedding, sectioning and staining of tissues.
2. Key steps include fixation of tissues using formalin to preserve structure, dehydration using increasing concentrations of alcohol, clearing with xylene, impregnation and embedding in paraffin wax.
3. Thin sections are then cut from the paraffin blocks using a microtome and stained, usually with hematoxylin and eosin, for microscopic examination.
Special Staining in Histopathology.pptxBhatyasir1234
This document describes the Verhoeff-van Gieson stain used to highlight elastic fibers in tissue sections. It stains elastic fibers black using Weigert's iron hematoxylin solution, and stains collagen red using Van Gieson solution. The stain allows differentiation of elastic fibers, collagen and other tissues under a microscope. It is commonly used because it produces intense staining of elastic fibers quickly.
This document discusses various staining techniques used in microbiology. It describes how stains work by binding to cellular components based on their electrical charge. Simple staining uses a single stain while differential staining compares how cells take up two contrasting stains. Gram staining is the most common differential technique, categorizing bacteria as Gram positive or negative based on their cell wall structure and ability to take up or release the primary stain. Numerous other specialized staining methods are described that target specific cellular structures like flagella, spores, or intracellular inclusions.
The document discusses the key steps in histological techniques which are used to prepare tissue samples for microscopic examination. The techniques include fixation, dehydration, clearing, embedding, sectioning, and staining of tissues. Fixation preserves the tissue structure using chemical fixatives. The tissue then undergoes dehydration, clearing and infiltration with paraffin wax before being embedded in wax blocks and sectioned for microscopic analysis. Staining helps to visualize different structures within the tissue sample under the microscope.
Histopathology is examination of tissues for presence or absence of changes in their structure due to disease processes. We go through various steps in the process of converting gross sample to microscopic slides.
1. The document discusses various cytological techniques used in the study and manipulation of cells including microtomy, microscopy, staining, and centrifugation.
2. Microtomy involves sectioning tissues into thin slices using a microtome for microscopic observation while staining allows cellular structures to be visualized through selective uptake of dyes.
3. Centrifugation separates particles according to properties like density, shape and size through application of centrifugal force, and is used to isolate cellular components through differential pelleting or density gradient centrifugation.
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1. The document discusses various cytological techniques used in the study and manipulation of cells including microtomy, microscopy, staining, and centrifugation.
2. Microtomy involves sectioning tissues into thin slices using a microtome for microscopic examination after fixation, dehydration, embedding, and staining.
3. Centrifugation separates particles in a solution based on properties like density, shape, and size through application of centrifugal force. Types include differential pelleting and density gradient centrifugation.
Histological specimen preparation involves fixing, processing, sectioning, and staining tissue samples. Key steps include:
1. Fixation prevents tissue degradation and preserves cellular structure. Common fixatives are formaldehyde and glutaraldehyde.
2. Tissue processing involves dehydration, clearing, and embedding tissues in paraffin wax to allow thin sectioning.
3. Sections are cut on a microtome and mounted on slides for staining. Staining with hematoxylin and eosin is most common, coloring nuclei blue and cytoplasm pink.
The major steps in tissue processing are dehydration, clearing, impregnation, and embedding. Dehydration removes water from tissues using increasing concentrations of alcohol. Clearing removes residual dehydrating agent using a substance miscible with both the dehydrating agent and paraffin wax. Impregnation completely replaces clearing agent with paraffin wax. Embedding involves orienting tissue in molten paraffin wax before it solidifies to provide structure for sectioning. Key advantages of this process are preservation of tissue structure and ability to cut thin, consistent sections for analysis.
This document discusses the process of tissue processing, which involves preparing tissue samples for microscopic examination through a series of reagents and embedding in a stable medium like paraffin wax. The key stages are fixation, dehydration, clearing, infiltration, and embedding. Factors that influence the quality of tissue processing include the choice of fixative and embedding medium, temperature, properties of processing reagents, tissue characteristics, and thickness. The goal is to minimize tissue damage and distortion during processing.
This document outlines the histotechnique process which tissues undergo before microscopic examination. Key steps include: fixation to preserve tissue structure; processing involving dehydration, clearing, and impregnation to allow sectioning; embedding tissues in paraffin blocks for microtomy; sectioning samples and staining, typically with hematoxylin and eosin, for visualization under the microscope. Finally, samples are mounted on slides and labeled for storage and pathological examination.
This document discusses tissue fixation techniques and histochemical staining methods. It begins by defining tissue fixation as a process that preserves tissues from decay through chemical means. It then describes various types of fixatives including simple fixatives like formalin and compound fixatives. Specific fixatives discussed include 10% neutral buffered formalin, Bouin's fluid, Zenker's fluid, and osmium tetroxide. Modes of action and uses of these fixatives are provided. The document concludes by outlining various histochemical staining techniques for identifying elements like glycogen, calcium, iron, uric acid crystals and amyloid. Specific stains discussed are PAS, Best's carmine, von Kossa's, Prussian blue, de Gal
This document discusses various staining techniques used to visualize cells and internal structures. It describes the basic components and principles of dyes and stains, including chromophores, auxochromes and benzene rings. It outlines different types of stains categorized by molecular structure and charge. Basic techniques like simple staining, gram staining and acid-fast staining are explained in detail. The document compares properties of gram-positive and gram-negative cell walls. It provides examples of structures that are acid-fast and the importance of Ziehl-Neelsen staining for detecting Mycobacterium tuberculosis bacilli.
This document discusses various staining techniques used to visualize cells and internal structures. It describes the basic components and principles of dyes and stains, including chromophores, auxochromes and benzene rings. It outlines different types of stains categorized by molecular structure and electric charge. Basic techniques like simple staining, gram staining and acid-fast staining are explained in detail. The document compares properties of gram-positive and gram-negative cell walls. It provides examples of structures that are acid-fast and the importance of Ziehl-Neelsen staining for detecting Mycobacterium tuberculosis bacilli.
This document discusses various staining techniques used in microscopy to visualize bacteria and other microscopic organisms. It describes different types of stains including simple stains that color all structures the same and differential stains that color different structures differently. Specific staining techniques are explained, including Gram staining to distinguish between Gram-positive and Gram-negative bacteria, acid-fast staining for mycobacteria, and endospore staining. The document provides details on procedures, requirements, and results for common staining methods.
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
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2. Tissue marking
Giving identification
(prevent loss of small
tissue)
Cutting a notch
Dye marking
( E.g. indian ink, Silver nitrate)
Criteria for selection of
tissue marker
Relatively insoluble
Can’t cause reagent conta
Remain on surface of
specimen
Clearly identifiable
4. Fixation
• Purpose is to preserve the
tissue structure
• First step in tissue preparation
• Aim:
arrest autolysis
Decomposition
Coagulate the tissue ( make
tissue life-like state)
Increase mechanical strength
• Fixatives can be classified
according to their;
1.Mechanism of action
2.Chemical nature
3.Combination
5. Determining fixation time of fixatives
• d= K x square root of T
Were d is depth of penetration
K is coefficient of diffusibility
T is period of immersion
6. Trimming
After fixation
To reach a sample size
Hard tissues (such as
bones and teeth) must
be decalcified before
trimming.
Decalcification
Complete removal of calcium
salt hard tissue following
fixation.
• Why decalcification?
1. Prevent tearing & ragging of
section
2. Prevent damage of microtome
knife/blades
3. Making bone soft
Achieved by:
• Dissolution of calcium by a
dilute mineral acid.
• Using Chelating agents EDTA
by eclectric current
13. Impregnation
• Make the tissue firm
• Facilitate easy sectioning
• 2 processes happen simultaneously
Impregnation of the paraffin wax in to tissue
Infiltration of clearing agent out of the tissue
• Also called internal embedding since medium penetrates
tissues & provides support from inside of tissue
• High melting point paraffin used
14. Embedding
• Gives external solid support
• Also called block preparation or external embeding
• Choice of paraffin depends on:
Nature of the tissue
Temp. at which tissues are sectioned
Thickness of the section
15. Question?
Q. 1. A senior lab technologist was performing
histopathological test in a histopathology lab. He
impregnated the dehydrated and cleared tissue with
molten paraffin wax before embedding. What
differentiates this impregnation process from
embedding?
A.Embedding for external support of tissue
B.Impregnation for external support of tissue
C.Impregnation is done by solid paraffin wax
D.Embedding is done by molten paraffin wax
18. Question?
• Q. 2. A fresh biopsy -5cm/hr was sent to a lab for
pathological examination. Lab tech. wanted to
make frozen section using cryostst. The cryostst
solidified the tissue in low temp.& produce
qualified thin tissue ribbons. Which activity can
the technologist perform by cryostat?
A. Dehydrating tissue with alcohols
B. Clearing tissue with clearing agents
C. Sectioning tissue to thin sections
D. Embedding tissue with paraffin medium
21. • Direct staining
Application of simple dye to
stain the tissue in varying
shades of colours
• Progressive staining
Stain applied to the tissue in
strict sequence and for
specific times.
• Regressive staining
Tissue is first overstained
and then the excess stain is
removed from all but the
structures to be
demonstrated. This process
is called differentiation
• Indirect staining
It means use of mordant to
facilitate a particular
staining method or the use
of accentuator to improve
either the selectivity or the
intensity of stain.
• Decolourization
Partial or complete removal
of stain from tissue sections.
E.g. acid alcohol treatment
22. Dyes used in staining
• Dyes are classified in various ways :
1. According to source (Natural & Synthetic)
2. Affinity to tissues (Acidophilic & Basophilic)
3. Chemical composition (Thiazines, Azo-dyes &
Rosailins)
23. Factors controlling selectivity of tissue component
Dyes or stains are not taken by every part of tissue
and this difference is called selectivity of stain
Factors that determine selectivity of stain include;
1. Number and affinity of binding sites
2. Rate of reagent uptake
3. Rate of reagent loss
4. Presence of non-dye constituent
Mordants and accentuators
23
24. • Mordants
Substance that causes certain staining reactions to
take place by forming a link between the tissue and
the stain. The link is referred as lake. e.g. Ammonium
and Potassium alum for haematoxylin.
• Accentuators
Substances that causes increase in selectively or in
staining power of dye. e.g. Phenol in Carbol fuchsin,
KOH in Mehtylene blue
25. Types of commonly used stains
Most staining reaction involve chemical interaction b/n dye and
substrate through salt linkage or hydrogen bonds
Staining with dyes result in predictable color pattern based on
acid/base characteristics of tissue and dye
Based on these stains can be divided in to
Acidic
Basic
Neutral
NB: This classification is not based on the pH of stains
25
26. Types………..
1. Acid dyes:
Exists as an anion in solution
has affinity for cytoplasm
• Stains the acidophilic structure
Most commonly used acidic stain is Eosin
26
27. Types………..
2. Basic dyes:
Exists as a cation in solution
has affinity for nuclei & ribosome
• Stains the basophilic structure
Most commonly used basic stain is Hematoxylin
27
28. Types………..
3. Neutral dye:
Combining aqueous solutions of acid and basic
dyes
Capable of staining cytoplasm and nucleus
simultaneously and differentially
– This is called methachromatic staining
e.g
• Giemsa’s stain
• leishman’s stain etc.
28
29. • Haematoxylin and Eosin staining
Procedure
♦ Deparaffinize in hot air oven.
♦ Hydrate the section.
i) 3 dips in xylene (2 Min. each)
ii) 3 dips in acetone / alcohol (2 Min. each)
iii) In running tap water for 5 Minutes.
♦ Mayer's haemotoxylin for 15 minutes.
♦ Wash in running tap water for 20 minutes
♦ Counter stain with eosin for 2 minutes
♦ Dehydrate the section in 95% and absolute alcohol/ acetone 2 changes (2minutes
each).
♦ Clear in xylene 3 changes (2 minutes each)
♦ Mount in DPX
29
30. Results of tissue staining
• Nucleus - blue
• Cytoplasm and background - pink
Causes of poor quality of staining
1. Poor or inadequate fixation of tissue.
2. Over or under-ripened Haematoxylin.
3. Overused or worked out Haematoxylin.
4. Over or under differentiation of haematoxylin
30
31. 5. Insufficient blueing following differentiation.
6. Failure to wash blueing agent out of section
before counter staining with eosin (especially
when ammonia is used).
7. Insufficient differentiation of eosin during
washing or dehydration.
8. Insufficient dehydration and clearing of sections.
9. Contamination of stains.
31
32. SPECIAL STAINING
VAN GIESON METHOD- staining of connective
tissue
• Principle: In the routine staining method collagen,
elastic fibres and smooth muscle appear pink or
reddish in colour.
• In the Van Gieson stain, collagen and most
reticulin stain selectively with acid aniline dyes
(acid fuchsin).
• Picric acid acts as counter stain for muscle and
cytoplasm and form complex with the dyes. This
complex has special affinity for collagen
32
33. Procedure
(1) Deparaffinize with xylene
(2) Hydration take sections to water
(3) Stain with Weigert’s haematoxylin for 20-40
minutes
(4) Wash in distilled water
(5) Van Gieson stain 1-3 min
(6) Rinse well in distilled water
(7) Dehydrate in absolute alcohol (2 changes)
(8) Clear in xylene (2 changes)
(9) Mount in DPX 33
34. • Results
1. Collagen Red
2. Muscle and Cornified epithelium -Yellow
(B) Nuclei -Blue to Black
34
35. McMANUS FOR GLYCOGEN (PAS)
Staining and identification of the various types of
carbohydrates
Principle : Tissue structures like liver & heart,
striated muscles are studied by Periodic acid Shiff
stain. Periodic acid reacts with aldehyde group of
the carbohydrates and afterwards reaction with
the schiff’s reagent produces a red or purple red
colour. 35
36. Procedure
1. Deparaffinize and hydrate to distilled water.
2. Oxidize in periodic acid solution for 5 minutes
3. Rinse in distilled water
4. Schiff’s regent solution for 15 minutes.
5. Wash in running water for 10 minutes for pink
colour to develop.
6. Harris haematoxylin for 6 minutes or light green
counter stain for a few seconds.
7. Wash in running water.
36
37. 8. Differentiate in 1% acid alcohol solution 3-10 quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue the sections
11. Wash in running water for 10 minutes
12. Dehydrate in 95% alcohol, absolute alcohol, clear in
xylene two changes each.
13. Mount in DPX.
37
38. • Results
With hematoxylin counterstain
1. Nuclei – blue
2. Glycogen, mucin, hyaluronic acid, reticulin, colloid
droplets, amyloid infiltration, thrombi. – purple
red
3. Fungi – Red
4. Background – pale green (with light green
counter staining).
38
39. Mounting of sections
After staining the section is mounted under a
cover slip using a syrup fluid
Why mounting?
1. To protect the specimen from physical injury
2. To protect the section from deterioration
3. To facilitate easy handling.
4. Preserving the slides for permanent storage
40. Mounting medium
A syrup fluid applied between the section and the cover slip
Characteristic of a good mounting medium
1. It should not dry quickly
2. It should not dissolve out or fade tissue sections
3. It should not cause shrinkage and distortion of tissues
4. It should set hard thereby producing permanent mounting of sections
5. RI should be as near as possible to that of the glass which is 1.518.
41. Mounting medium……….
There are two types of mounting media
1.Aqueous media
2.Resinous media
Aqueous media
designed to mount water miscible preparations
I. Glycerin –RI is 1.47
II. Farrant’s medium –RI of 1.43
III. Apathy’s medium –RI of 1.52
42. Mounting medium……….
Resinous media
For preparations that have been dehydrated
Sections cleared by Toludine/ xylene
1. Canada balsam - Canadian tree Abus Balsamea.
• Mount most type of tissue sections and also for thicker tissues
• refractive index is 1.54
2. DPX - for small tissue sections
• A mixture of distyrene (polystyrene), plasticizer (tricresyl
phosphate), and xylene
• refractive index is 1.53
3.XAM
• synthetic resin mixed with Xylene
• Its refractive index is 1.52.
43. Mounting
Make quite sure the sections are clear, do not let the section go dry before
mounting
1. Hold the slide b/n thumb and forefinger and wipe both ends of the slid
2. Clean carefully around section and lay on a clean blotting paper with
section uppermost with appropriate cover slip
3. Place a drop of mountant on the cover slip
4. Invert the slide over the cover slip and lower it so that it just adhere to
the cover slip
43
44. Mounting…………
5. Quickly turn slide over, then lay it on flat
surface to allow the mountant to spread
6. Wipe around the covers lip for neatness
N.B:
There should be no air bubbles, if any it should be returned to xylene to
remove cover slip
No attempt should be made to pull the cover slip
Slight warming of the slide from below will make small air bubbles to
escape.
44
45. • When preparing a tissue for Histopathology
and light microscopy , which method precedes
clearing the specimen with an organic solvent,
a. Fixation
b. Clearing
c. Staining
d. Dehydration
e. Embeding
Editor's Notes
n optics the refractive index or index of refraction n of an optical medium is a dimensionless number that describes how light, or any other radiation, propagates through that medium. It is defined as
n=c/v;ll,lmlhjknmnmnmnmmnmknooo;p;p;;p[‘[[‘[
where c is the speed of light in vacuum and v is the phase velocity of light in the medium
Canada balsam, also called Canada turpentine or balsam of fir, is a turpentine which is made from the resin of the balsam fir tree (Abies balsamea) of boreal North America. The resin, dissolved in essential oils, is a viscous, sticky, colourless or yellowish liquid that turns to a transparent yellowish mass when the essential oils have been allowed to evaporate.
Canada balsam is amorphous when dried. Since it does not crystallize with age, its optical properties do not deteriorate.[citation needed] However, it has poor thermal and solvent resistance.[1]
Resin, in the most specific meaning of the term, is a hydrocarbon secretion of many plants, particularly coniferous trees. It is distinct from other liquid compounds found inside plants or exuded by plants, such as sap, latex, or mucilage. More broadly, the term "resin" is also used for many thick liquids, some of them artificial polymer bases (synthetic resins), that during normal use, harden into transparent or opaque solids
DPX and BPS Synthetic Resin Mountants
A mixture of distyrene (a polystyrene), a plasticizer (tricresyl phosphate), and xylene, called DPX, was introduced in 1939 and later modified by the substitution of a more satisfactory plasticizer, dibutylphthalate (butyl, phthalate, styrene - BPS). This colorless, synthetic resin mounting media is now available at Electron Microscopy Sciences, DPX, and it has generally replaced xylene-balsam. They preserve stains and dry quickly; surplus mount
ant may be peeled off the preparation
after cutting around the cover slip with a razor bl
ade or scalpel. They are not recommended
for use with thick sections (eg. cellulose nitrate)
where there is a danger of retraction of the
mountant upon drying.
Refractive index ( 15 C ) 1.525
Canada balsam
,
yellow, oily, resinous exudation obtained from the
balsam fir
. It is an
oleoresin
with a pleasant odor but a biting taste. It is turp
entine rather than a true balsam.
On standing, the essential oil in Canada balsam eva
porates, leaving behind the resin as a
hard, transparent varnish. Canada balsam is valued
as an optical mounting cement, e.g., for
lenses and microscope slides, since it yields, when
dissolved in an equal volume of xylene, a
noncrystallizing cement with a refractive index nea
rly equal to that of ordinary glass. It is
used also in paints and polishes.
Canada balsam, also called Canada turpentine or bal
sam of fir, is a turpentine which is
made from the resin of the balsam fir tree (Abies b
alsamea).
It is the fir's resin, dissolved in essential oils,
and is a viscous, sticky, colourless (sometimes
yellowish) liquid, that turns to a transparent yell
owish mass when the essential oils have
been allowed to evaporate.
Due to its high optical quality, its
refractive index (n = 1.55, very close to that of g
lass),
and
its purity it is mainly used in optics as an invisi
ble-when-dry glue for glass. It is soluble in
xylene, amorphous when dried, and it does not cryst
allize with age, so its optical properties
do not deteriorate.[citation needed]