This document provides instructions for several histopathology staining techniques, including:
- Periodic Acid Schiff (PAS) staining for polysaccharides and basement membranes in magenta.
- Gram-Twort modification for staining bacteria in paraffin sections in blue-black (Gram positive) and pink (Gram negative).
- Ziehl-Neelsen technique for staining acid-fast bacilli like Mycobacterium tuberculosis red against a blue background.
It also describes the Periodic Acid Schiff/Alcian Blue dual stain to differentiate acid mucins (blue) from neutral mucins and carbohydrates (magenta). Precise protocols and reagent preparations are provided for accurate histological analysis.
This document discusses cytology of the urinary tract. It describes three methods for collecting urine specimens: voided urine, catheter specimens, and bladder washings. Voided urine is the simplest but cells may degrade over time. Catheter specimens avoid contamination but can damage cells. Bladder washings provide high cellularity and preservation through saline irrigation. Specimens can be prepared via several methods including cytocentrifugation and direct smears. Normal urinary tract cytology shows a range of superficial and deep urothelial cell morphologies depending on collection method and location in the tract.
Frozen section is a pathology procedure that allows rapid microscopic examination of a specimen during surgery. Sir Louis B. Wilson pioneered the technique in 1905 at the Mayo Clinic to enable urgent intraoperative diagnosis. The procedure involves snap freezing tissue, sectioning it with a cryostat microtome, and staining for quick analysis. While fast, frozen sections can have artifacts from ice crystals and knife marks. Pathologists must communicate closely with surgeons to ensure the appropriate use of frozen sections for urgent diagnostic needs during operations.
The document discusses cell blocks, which are used in cytopathology to provide tissue samples from fluid specimens for histological examination. Cell blocks allow for maintaining tissue architecture, performing ancillary tests, and archiving samples. Various cell block preparation methods are described. Cell blocks provide diagnostic advantages over smears for certain tumor types and body fluids. While cell blocks increase diagnostic accuracy, some methods can result in low cellularity or inadequate samples for ancillary testing. Overall, the document provides an overview of the utility and methods of cell block preparation in cytopathology.
This document discusses various staining techniques used in cytology, including both routine and special stains. It provides details on the principles, procedures, and applications of stains such as May-Grunwald Giemsa, Diff-Quik, Papanicolaou, hematoxylin and eosin, periodic acid Schiff, mucicarmine, Alcian blue, Oil red O, Congo red, Feulgen, and Ziehl-Neelsen. The stains are used to demonstrate cellular and extracellular components, identify infectious organisms, and examine DNA content. Proper staining allows visualization of structures like glycogen, mucin, lipids, amyloid, and acid-fast bacteria under the microscope.
COMPARISON OF CONVENTIONAL PAPANICOLAOU STAIN WITH MODIFIED ULTRAFAST PAPANIC...SURAMYA BABU
• Body fluid cytology is vital in diagnosis of various neoplastic and non neoplastic lesions and conventional Pap stain is the staining method of choice for the same.
• MUFP is a quick and cheap staining technique which gives good interpretation of cytological features with easily available reagents.
• Preservation of cell morphology and nuclear staining are superior with conventional Papanicolaou technique whereas cytoplasmic staining is comparable with conventional pap and MUFP techniques.
• Though background of stained smears was slightly better with conventional Pap staining; MUFP was superior in case of hemorrhagic samples.
Cytopathology is the study of exfoliated cells to detect normal and abnormal tissue morphology. Cells can be collected naturally or artificially from various body sites like skin, cervix, lungs, and lymph nodes. Cytopathology allows rapid, inexpensive diagnosis and monitoring of diseases without surgery. Pap smears screen for cervical cancer by examining cells from the cervix and vagina. Abnormal findings on Pap smears require follow up with colposcopy and possible biopsy. Screening reduces cervical cancer rates by facilitating early detection and treatment of precancerous lesions.
George Papanicolaou is considered the father of cytopathology. He developed the Pap test in the 1920s as a screening method for cervical cancer. The Pap test involves collecting cells from the cervix and examining them under a microscope. It allows for early detection of precancerous and cancerous cells. Though initially met with skepticism, the Pap smear became widely accepted in the 1940s and has significantly reduced cervical cancer mortality rates. The test involves collecting a cell sample, processing and staining the cells, then examining them under a microscope to look for abnormalities. It is a simple yet effective screening tool.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
This document discusses cytology of the urinary tract. It describes three methods for collecting urine specimens: voided urine, catheter specimens, and bladder washings. Voided urine is the simplest but cells may degrade over time. Catheter specimens avoid contamination but can damage cells. Bladder washings provide high cellularity and preservation through saline irrigation. Specimens can be prepared via several methods including cytocentrifugation and direct smears. Normal urinary tract cytology shows a range of superficial and deep urothelial cell morphologies depending on collection method and location in the tract.
Frozen section is a pathology procedure that allows rapid microscopic examination of a specimen during surgery. Sir Louis B. Wilson pioneered the technique in 1905 at the Mayo Clinic to enable urgent intraoperative diagnosis. The procedure involves snap freezing tissue, sectioning it with a cryostat microtome, and staining for quick analysis. While fast, frozen sections can have artifacts from ice crystals and knife marks. Pathologists must communicate closely with surgeons to ensure the appropriate use of frozen sections for urgent diagnostic needs during operations.
The document discusses cell blocks, which are used in cytopathology to provide tissue samples from fluid specimens for histological examination. Cell blocks allow for maintaining tissue architecture, performing ancillary tests, and archiving samples. Various cell block preparation methods are described. Cell blocks provide diagnostic advantages over smears for certain tumor types and body fluids. While cell blocks increase diagnostic accuracy, some methods can result in low cellularity or inadequate samples for ancillary testing. Overall, the document provides an overview of the utility and methods of cell block preparation in cytopathology.
This document discusses various staining techniques used in cytology, including both routine and special stains. It provides details on the principles, procedures, and applications of stains such as May-Grunwald Giemsa, Diff-Quik, Papanicolaou, hematoxylin and eosin, periodic acid Schiff, mucicarmine, Alcian blue, Oil red O, Congo red, Feulgen, and Ziehl-Neelsen. The stains are used to demonstrate cellular and extracellular components, identify infectious organisms, and examine DNA content. Proper staining allows visualization of structures like glycogen, mucin, lipids, amyloid, and acid-fast bacteria under the microscope.
COMPARISON OF CONVENTIONAL PAPANICOLAOU STAIN WITH MODIFIED ULTRAFAST PAPANIC...SURAMYA BABU
• Body fluid cytology is vital in diagnosis of various neoplastic and non neoplastic lesions and conventional Pap stain is the staining method of choice for the same.
• MUFP is a quick and cheap staining technique which gives good interpretation of cytological features with easily available reagents.
• Preservation of cell morphology and nuclear staining are superior with conventional Papanicolaou technique whereas cytoplasmic staining is comparable with conventional pap and MUFP techniques.
• Though background of stained smears was slightly better with conventional Pap staining; MUFP was superior in case of hemorrhagic samples.
Cytopathology is the study of exfoliated cells to detect normal and abnormal tissue morphology. Cells can be collected naturally or artificially from various body sites like skin, cervix, lungs, and lymph nodes. Cytopathology allows rapid, inexpensive diagnosis and monitoring of diseases without surgery. Pap smears screen for cervical cancer by examining cells from the cervix and vagina. Abnormal findings on Pap smears require follow up with colposcopy and possible biopsy. Screening reduces cervical cancer rates by facilitating early detection and treatment of precancerous lesions.
George Papanicolaou is considered the father of cytopathology. He developed the Pap test in the 1920s as a screening method for cervical cancer. The Pap test involves collecting cells from the cervix and examining them under a microscope. It allows for early detection of precancerous and cancerous cells. Though initially met with skepticism, the Pap smear became widely accepted in the 1940s and has significantly reduced cervical cancer mortality rates. The test involves collecting a cell sample, processing and staining the cells, then examining them under a microscope to look for abnormalities. It is a simple yet effective screening tool.
This document discusses decalcification, which is the process of removing calcium from bone and other calcified tissues prior to sectioning and microscopic examination. It defines decalcification and lists the criteria for an ideal decalcifying agent. Various factors that affect the rate of decalcification are described, including concentration, temperature, agitation, and suspension of the tissue. The main methods of decalcification are outlined as well as the principles, types, compositions, and procedures for different decalcifying agents such as acids, ion exchange resins, and chelating agents.
The document discusses Pap smear testing, including:
- The definition and purpose of the Pap smear test in screening for cervical cancer.
- Guidelines for patient preparation, specimen collection using an Ayres spatula and cytobrush, sample preparation including fixation and staining.
- Criteria for evaluating specimen adequacy, including what constitutes an adequate, satisfactory but limited, and unsatisfactory smear.
- What constitutes normal cervical cells and abnormalities that may be detected, following the Bethesda system for reporting cervical cytology.
This document discusses the process of histology, which involves preparing tissue samples for microscopic examination. Specimens should be transported in fixatives like formalin or refrigerated. There are several steps involved including fixation, processing, sectioning, staining, and mounting. Tissue processors are used to dehydrate samples through a series of alcohols and xylene before infiltrating and embedding them in paraffin wax for sectioning with a microtome. Sections are then stained using techniques like H&E staining before examination under a microscope. Frozen sectioning and newer techniques using resins and microwaves can also be used to prepare samples for histological analysis.
The document discusses cryostats, which are devices used to cut thin frozen sections of tissues for examination under a microscope. Cryostats contain a microtome inside a freezer unit that can rapidly freeze tissue samples and cut sections as thin as 1 micrometer at temperatures below freezing. The cryostat process allows for quick diagnosis by freezing and sectioning tissues within minutes rather than having to dehydrate, embed in paraffin, and section as with traditional microtomes.
This document discusses cytopreparatory techniques, including fixation of cytological samples, staining methods, and interpretation. It focuses on fixation, explaining that fixation preserves cells in a lifelike state after death by preventing autolysis and putrefaction. The key properties of a good fixative are outlined, and various fixatives are classified and examples are provided, including alcohols, formalin, and mercuric chloride, which are commonly used for cytological preparations.
This document describes the steps involved in tissue processing from fixation to embedding in wax. It discusses obtaining fresh specimens, fixation in formalin, dehydration through an alcohol series, clearing in xylene, infiltration and embedding in paraffin wax. Sections are then cut on a microtome, mounted on slides and stained, usually with hematoxylin and eosin, to visualize tissue structures microscopically. Proper processing is important to preserve tissue morphology and produce high quality stained sections for diagnostic examination.
This document provides an overview of tissue fixation techniques. It defines fixation as a process that preserves tissues in a state close to how they appeared when living. This is achieved by preventing autolysis and maintaining cellular morphology. The document discusses various types of fixatives including aldehydes, alcohols, and oxidizing agents. It also covers the aims of fixation, how different fixatives work, commonly used fixatives for different tissue and cellular components, and potential artifacts. Fixation is essential for histological examination and aims to maintain tissues for further analysis.
This document provides information on immunohistochemistry (IHC), including:
1. IHC is used to detect antigens in tissues through antigen-antibody recognition at the light microscopic level. It applies immunologic principles and techniques to study cells and tissues.
2. The basic principle of IHC is a sharp visualization of target components in cells and tissues based on a satisfactory signal-to-noise ratio.
3. The main steps of IHC are tissue processing, antigen retrieval, primary/secondary antibody incubation, detection, counterstaining, and mounting. Proper controls and interpretation of results are also discussed.
Cell blocks provide diagnostic information in addition to regular cytology slides. They allow examination of histological structure and use of ancillary tests like special stains and immunohistochemistry. A cell block is prepared by concentrating cells from cytology specimens using various methods like centrifugation or thrombin clotting. This allows cells to be processed and examined like histology samples. Cell blocks improve diagnostic accuracy for body fluids and fineneedle aspiration samples. They are useful for identifying primary tumor sites, distinguishing reactive from malignant cells, and enabling molecular testing.
The frozen section procedure allows for rapid microscopic analysis of tissue specimens. Tissue is frozen to preserve its structure and cellular components. This allows thin sections of the tissue to be cut without chemical processing. Frozen sections are used for rapid diagnosis during surgery and research applications where chemical fixation could damage antigens or enzymes. The cryostat machine houses a microtome that can precisely cut thin frozen sections at controlled temperatures for examination.
Washed red blood cell suspensions are prepared to remove plasma proteins that could interfere with antigen-antibody reactions during blood typing tests. The red blood cells are separated from whole blood via centrifugation and washed with saline to remove plasma. This helps remove soluble antigens, interfering proteins, and substances that could cause false positive reactions. The washed red blood cells are then suspended in saline at a 3-5% concentration for use in blood typing tests.
This document discusses tissue processing and fixation. It begins by introducing tissue fixation and its objectives, such as preventing degradation and maintaining morphology. It then describes various fixation methods and factors that affect fixation quality. Common fixatives are discussed, including formaldehyde, glutaraldehyde, Zenker's solution and Bouin's solution. Fixation protocols for specific tissues like brain, breast, lung and kidney are also reviewed. The document emphasizes the importance of proper fixation for histological examination.
This document describes various smear preparation techniques used in cytology, including direct smears, blood smear technique, squash technique, large volume centrifugation, small volume centrifugation, membrane filtration, cell blocks, density gradient centrifugation, and gravity sedimentation. Direct smears involve spreading the specimen directly onto a slide. Blood smear technique produces a thin, uniform smear for staining. Squash technique results in a thin, uniform preparation. Large volume centrifugation concentrates fluid specimens by separating the buffy coat layer. Small volume centrifugation uses a cyto-centrifuge to deposit cells directly onto a slide. Membrane filtration uses a filter to collect cells on a slide. Cell blocks allow processing samples as histopath
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.
Decalcification is a process used to remove mineral content from calcified tissues like bone and teeth to allow for microscopic examination. It involves selecting an appropriate decalcifying agent based on factors like the tissue, required staining, and urgency. Common decalcifying agents include acids like nitric acid, formic acid, and chelating agents. The decalcification process must be monitored and the tissue properly processed, sectioned, and stained afterwards to examine it microscopically. Undecalcified sections can also be prepared to examine mineralized and non-mineralized bone ratios.
Staining ( rouine and special in cytology) rajiv kumarrajusehrawat
The document discusses staining techniques used in histology and cytology. It provides details on the preparation, components, and use of common stains including Hematoxylin, Giemsa stain, Papanicolaou stain, and Periodic acid–Schiff stain. The stains are used to differentially color structures like nuclei, cytoplasm, muscles, bones, parasites and glycogen under the microscope to enable examination of tissue samples and identification of cells and microorganisms.
diagnostic Cytology introduction , Body fluids cytologyAayra
This document discusses diagnostic cytopathology. It covers:
1. Cytopathology examines cells from body cavities, mucosal surfaces, and organs/masses obtained via needle aspiration to determine the cause of disease microscopically.
2. The history of cytopathology including the contributions of Papanicolaou and Koss.
3. The advantages of cytopathology include rapid diagnosis, low cost, ability to sample without tissue injury, and ability to repeatedly sample. Disadvantages include inability to always determine tumor type or distinguish pre-invasive from invasive changes.
4. Types of cytopathology include exfoliative from spontaneously shed cells, abrasive which dislodges
Embedding is the process of enclosing tissue specimens in an embedding medium such as paraffin wax to support the specimen for sectioning. The choice of embedding medium depends on the type of tissue, microscope, and microtome being used. Common embedding mediums include paraffin wax, celloidin, resin, and gelatin. Paraffin wax is most widely used due to its hardness and ability to produce high quality sections. Proper orientation of the specimen in the embedding block is important for pathological examination and diagnosis.
CYTOLOGY AND CYTOGENITICS MCQS BY IKRAM ULLAH Ikram Ullah
This document contains 60 multiple choice questions about cytology and cytogenetics. It covers topics like the structures and functions of cells and their organelles, cell membrane models, the role of mitochondria and lysosomes, fixation techniques used in cytology including different fixatives and their properties, staining methods like Papanicolaou staining, and normal cytology of organs like the uterus and cervix. The questions are multiple choice with a single correct answer for each question.
This document provides information on various histopathology staining techniques. It describes the steps for taking paraffin sections to water, dehydrating and clearing sections in xylene, blotting sections dry, and mounting sections. It also details procedures for Ziehl-Neelsen staining for acid-fast bacilli, Gram-Twort staining for bacteria, Periodic acid Schiff staining, Periodic acid Schiff/Alcian blue staining, and the buffered Congo red method for amyloid. Precise reagents and safety notes are included for each technique.
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.
The document discusses Pap smear testing, including:
- The definition and purpose of the Pap smear test in screening for cervical cancer.
- Guidelines for patient preparation, specimen collection using an Ayres spatula and cytobrush, sample preparation including fixation and staining.
- Criteria for evaluating specimen adequacy, including what constitutes an adequate, satisfactory but limited, and unsatisfactory smear.
- What constitutes normal cervical cells and abnormalities that may be detected, following the Bethesda system for reporting cervical cytology.
This document discusses the process of histology, which involves preparing tissue samples for microscopic examination. Specimens should be transported in fixatives like formalin or refrigerated. There are several steps involved including fixation, processing, sectioning, staining, and mounting. Tissue processors are used to dehydrate samples through a series of alcohols and xylene before infiltrating and embedding them in paraffin wax for sectioning with a microtome. Sections are then stained using techniques like H&E staining before examination under a microscope. Frozen sectioning and newer techniques using resins and microwaves can also be used to prepare samples for histological analysis.
The document discusses cryostats, which are devices used to cut thin frozen sections of tissues for examination under a microscope. Cryostats contain a microtome inside a freezer unit that can rapidly freeze tissue samples and cut sections as thin as 1 micrometer at temperatures below freezing. The cryostat process allows for quick diagnosis by freezing and sectioning tissues within minutes rather than having to dehydrate, embed in paraffin, and section as with traditional microtomes.
This document discusses cytopreparatory techniques, including fixation of cytological samples, staining methods, and interpretation. It focuses on fixation, explaining that fixation preserves cells in a lifelike state after death by preventing autolysis and putrefaction. The key properties of a good fixative are outlined, and various fixatives are classified and examples are provided, including alcohols, formalin, and mercuric chloride, which are commonly used for cytological preparations.
This document describes the steps involved in tissue processing from fixation to embedding in wax. It discusses obtaining fresh specimens, fixation in formalin, dehydration through an alcohol series, clearing in xylene, infiltration and embedding in paraffin wax. Sections are then cut on a microtome, mounted on slides and stained, usually with hematoxylin and eosin, to visualize tissue structures microscopically. Proper processing is important to preserve tissue morphology and produce high quality stained sections for diagnostic examination.
This document provides an overview of tissue fixation techniques. It defines fixation as a process that preserves tissues in a state close to how they appeared when living. This is achieved by preventing autolysis and maintaining cellular morphology. The document discusses various types of fixatives including aldehydes, alcohols, and oxidizing agents. It also covers the aims of fixation, how different fixatives work, commonly used fixatives for different tissue and cellular components, and potential artifacts. Fixation is essential for histological examination and aims to maintain tissues for further analysis.
This document provides information on immunohistochemistry (IHC), including:
1. IHC is used to detect antigens in tissues through antigen-antibody recognition at the light microscopic level. It applies immunologic principles and techniques to study cells and tissues.
2. The basic principle of IHC is a sharp visualization of target components in cells and tissues based on a satisfactory signal-to-noise ratio.
3. The main steps of IHC are tissue processing, antigen retrieval, primary/secondary antibody incubation, detection, counterstaining, and mounting. Proper controls and interpretation of results are also discussed.
Cell blocks provide diagnostic information in addition to regular cytology slides. They allow examination of histological structure and use of ancillary tests like special stains and immunohistochemistry. A cell block is prepared by concentrating cells from cytology specimens using various methods like centrifugation or thrombin clotting. This allows cells to be processed and examined like histology samples. Cell blocks improve diagnostic accuracy for body fluids and fineneedle aspiration samples. They are useful for identifying primary tumor sites, distinguishing reactive from malignant cells, and enabling molecular testing.
The frozen section procedure allows for rapid microscopic analysis of tissue specimens. Tissue is frozen to preserve its structure and cellular components. This allows thin sections of the tissue to be cut without chemical processing. Frozen sections are used for rapid diagnosis during surgery and research applications where chemical fixation could damage antigens or enzymes. The cryostat machine houses a microtome that can precisely cut thin frozen sections at controlled temperatures for examination.
Washed red blood cell suspensions are prepared to remove plasma proteins that could interfere with antigen-antibody reactions during blood typing tests. The red blood cells are separated from whole blood via centrifugation and washed with saline to remove plasma. This helps remove soluble antigens, interfering proteins, and substances that could cause false positive reactions. The washed red blood cells are then suspended in saline at a 3-5% concentration for use in blood typing tests.
This document discusses tissue processing and fixation. It begins by introducing tissue fixation and its objectives, such as preventing degradation and maintaining morphology. It then describes various fixation methods and factors that affect fixation quality. Common fixatives are discussed, including formaldehyde, glutaraldehyde, Zenker's solution and Bouin's solution. Fixation protocols for specific tissues like brain, breast, lung and kidney are also reviewed. The document emphasizes the importance of proper fixation for histological examination.
This document describes various smear preparation techniques used in cytology, including direct smears, blood smear technique, squash technique, large volume centrifugation, small volume centrifugation, membrane filtration, cell blocks, density gradient centrifugation, and gravity sedimentation. Direct smears involve spreading the specimen directly onto a slide. Blood smear technique produces a thin, uniform smear for staining. Squash technique results in a thin, uniform preparation. Large volume centrifugation concentrates fluid specimens by separating the buffy coat layer. Small volume centrifugation uses a cyto-centrifuge to deposit cells directly onto a slide. Membrane filtration uses a filter to collect cells on a slide. Cell blocks allow processing samples as histopath
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.
Decalcification is a process used to remove mineral content from calcified tissues like bone and teeth to allow for microscopic examination. It involves selecting an appropriate decalcifying agent based on factors like the tissue, required staining, and urgency. Common decalcifying agents include acids like nitric acid, formic acid, and chelating agents. The decalcification process must be monitored and the tissue properly processed, sectioned, and stained afterwards to examine it microscopically. Undecalcified sections can also be prepared to examine mineralized and non-mineralized bone ratios.
Staining ( rouine and special in cytology) rajiv kumarrajusehrawat
The document discusses staining techniques used in histology and cytology. It provides details on the preparation, components, and use of common stains including Hematoxylin, Giemsa stain, Papanicolaou stain, and Periodic acid–Schiff stain. The stains are used to differentially color structures like nuclei, cytoplasm, muscles, bones, parasites and glycogen under the microscope to enable examination of tissue samples and identification of cells and microorganisms.
diagnostic Cytology introduction , Body fluids cytologyAayra
This document discusses diagnostic cytopathology. It covers:
1. Cytopathology examines cells from body cavities, mucosal surfaces, and organs/masses obtained via needle aspiration to determine the cause of disease microscopically.
2. The history of cytopathology including the contributions of Papanicolaou and Koss.
3. The advantages of cytopathology include rapid diagnosis, low cost, ability to sample without tissue injury, and ability to repeatedly sample. Disadvantages include inability to always determine tumor type or distinguish pre-invasive from invasive changes.
4. Types of cytopathology include exfoliative from spontaneously shed cells, abrasive which dislodges
Embedding is the process of enclosing tissue specimens in an embedding medium such as paraffin wax to support the specimen for sectioning. The choice of embedding medium depends on the type of tissue, microscope, and microtome being used. Common embedding mediums include paraffin wax, celloidin, resin, and gelatin. Paraffin wax is most widely used due to its hardness and ability to produce high quality sections. Proper orientation of the specimen in the embedding block is important for pathological examination and diagnosis.
CYTOLOGY AND CYTOGENITICS MCQS BY IKRAM ULLAH Ikram Ullah
This document contains 60 multiple choice questions about cytology and cytogenetics. It covers topics like the structures and functions of cells and their organelles, cell membrane models, the role of mitochondria and lysosomes, fixation techniques used in cytology including different fixatives and their properties, staining methods like Papanicolaou staining, and normal cytology of organs like the uterus and cervix. The questions are multiple choice with a single correct answer for each question.
This document provides information on various histopathology staining techniques. It describes the steps for taking paraffin sections to water, dehydrating and clearing sections in xylene, blotting sections dry, and mounting sections. It also details procedures for Ziehl-Neelsen staining for acid-fast bacilli, Gram-Twort staining for bacteria, Periodic acid Schiff staining, Periodic acid Schiff/Alcian blue staining, and the buffered Congo red method for amyloid. Precise reagents and safety notes are included for each technique.
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.
This document discusses various special staining methods used in bacteriology. It describes acid-fast staining including Ziehl-Neelsen technique. Other staining methods discussed are fluorochrome staining for acid-fast bacilli, spore staining including Schaeffer-Fulton method, capsule staining using positive and negative techniques, flagella staining using Ryu's stain, lipid staining with Burdon's and Holbrook & Anderson methods, and Romanowsky staining techniques like Giemsa stain. The document provides detailed procedures for these special staining methods used to identify different bacterial structures under the microscope.
This document discusses various special staining methods used in bacteriology. It describes acid-fast staining including Ziehl-Neelsen technique. Other staining methods discussed are fluorochrome staining for acid-fast bacilli, spore staining including Schaeffer-Fulton method, capsule staining using positive and negative techniques, flagella staining using Ryu's stain, lipid staining with Burdon's and Holbrook & Anderson methods, and spirochete staining with Fontana's silver impregnation. It also discusses Romanowsky staining techniques like Giemsa stain and acridine orange staining for nucleic acids under UV light.
Plastination is a process for long-term preservation of biological tissues that leaves them completely dry but still life-like. It involves removing water and lipids from tissues and replacing them with curable polymers like silicone or epoxy. There are three main steps - fixation in formalin, dehydration in acetone, and forced impregnation with polymer under vacuum. The polymer is then hardened to complete plastination. Whole bodies, organs, or thin cross-sectional sheets can be plastinated. Plastinated specimens are durable, pose no health risks, and are useful for teaching anatomy. However, plastination is an expensive and time-consuming process requiring specialized equipment and skills.
This document discusses cytogenetics and chromosome analysis techniques. It begins with an introduction to human chromosomes and chromosomal abnormalities. It then describes various types of chromosomal mutations and abnormalities that can be detected through karyotyping and fluorescence in situ hybridization (FISH). The document provides detailed procedures for chromosome sample preparation from bone marrow and blood cultures, as well as staining and analysis techniques like Giemsa staining and G-banding. The importance of chromosomal studies for diagnosing conditions like Turner syndrome and Klinefelter syndrome is also highlighted.
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.
The document discusses various techniques used in histopathology sample processing including decalcification, fixation, dehydration, clearing, embedding and sectioning. It covers different chemical agents used for each step along with their properties and advantages. Various methods are described such as paraffin, celloidin and vacuum embedding for optimal tissue preservation and section quality. Automatic tissue processors and freeze drying are also mentioned as techniques to reduce processing time.
The document discusses the process of tissue processing and embedding tissue samples in paraffin wax blocks. It explains that tissue processing involves dehydration to remove water, clearing to remove dehydrating agents, and infiltration/impregnation to replace clearing agents with molten paraffin wax. The aim is to embed tissues in a solid medium that supports sectioning. Key steps include using increasing concentrations of alcohol for dehydration, xylene or toluene for clearing due to their miscibility with dehydrating agents and paraffin, and replacing clearing agents with molten paraffin wax of varying hardness during infiltration. This embeds tissues in paraffin wax blocks to make thin sections for histological examination.
Each chromosome in the somatic-cell complement can be uniquely identified by following a number of different banding procedures.
The banding patterns are highly characteristic. The International System for Cytogenetic Nomenclature (ISCN) provides schematic representations, or Ideograms, of human chromosomes corresponding to approx. 400, 550, and 850 bands per haploid set (I).
Although under constant revision, its principles rest on a numbering system based on major bands as they appear from the centromere outward along each chromosome arm.
To the cytogeneticist, the appearance of well-prepared, clearly banded chromosomes has an aesthetic appeal that is often difficult for the non-cytogeneticist to comprehend.
This document provides information about lab exercises on gram staining, endospore staining, and capsule staining. It includes the procedures for each stain and discusses what structures each stain targets (e.g. gram stain targets the peptidoglycan cell wall). It also provides background on Bacillus anthracis and how it can cause disease. Key points covered are that the gram stain differentiates bacteria types, endospore stain uses malachite green to stain spores, and the capsule stain demonstrates capsules using Congo red and Maneval's solution.
This document provides an overview of tissue processing techniques used in histology and histopathology. It discusses the various steps involved, including dehydration, clearing, infiltration, and embedding in paraffin wax or other mediums. It describes the purposes and methods for dehydration, clearing using agents like xylene, infiltration using paraffin wax, and embedding tissues. It also discusses alternative embedding techniques like ester wax, water soluble waxes, gelatin, and celloidin embedding as well as double embedding methods and potential processing artifacts.
This document discusses various staining techniques used to identify microorganisms under a microscope. It describes staining methods such as Gram staining, acid-fast staining, and Leoffler's methylene blue staining. These staining methods are used to differentiate bacteria and examine their morphology, size, and arrangement. The document also discusses the types of microscopes used such as light and electron microscopes.
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.
This document describes the synthesis of adipic acid from cyclohexanone via a nitric acid oxidation reaction. The reaction is exothermic and the nitric acid must be added slowly to cyclohexanone. Once complete, the product crystallizes and is washed and dried. The percent yield was 62.33% and melting point was 148-151°C, slightly lower than literature due to residual solvent. Infrared spectroscopy confirmed the product was adipic acid. Safety precautions are described for handling oxidizing and acidic reagents.
Tissue processing involves fixing, dehydrating, clearing, and infiltrating tissue samples with paraffin wax to embed them for sectioning. The key steps are fixation to prevent degradation, dehydration using graded alcohols, clearing with solvents like xylene to remove alcohol, infiltration using paraffin wax, embedding wax blocks for sectioning, sectioning on a microtome, and staining for examination. Automated tissue processors can complete many processing steps unattended for increased efficiency and throughput in pathology laboratories. Proper handling and processing is essential to obtain an accurate histological diagnosis from tissue specimens.
This document discusses various staining methods for acid-fast bacteria, including the Ziehl-Neelsen stain and modifications such as Kinyoun's stain, cold stains like Gabbett's method, and stains for tissue sections or spores. Key methods mentioned are Kinyoun's stain, which does not require heating; Gabbett's cold stain which is a two-step method for discolorization and counterstaining; and stains for tissue sections like Fite-Faraco, Fite, and Wade Fite stains which use special treatments to preserve the delicate cell walls of bacteria like M. leprae.
Students gathered for a session to discuss their studies and plans. Many shared challenges they faced with keeping up with their coursework while balancing other responsibilities. Ideas were exchanged on effective time management strategies and using campus resources to stay on track academically.
This document provides information about lipid profiles and their normal ranges. It discusses how to collect and process blood specimens for lipid profiles. It then defines the components of a lipid profile including total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, and VLDL cholesterol. For each component, it provides the normal ranges and clinical significance of abnormal levels. It also briefly discusses apolipoprotein B, phospholipids, chylomicrons, and factors that can increase or decrease their levels.
Carbohydrates, lipids, proteins, and nucleic acids are the four major macromolecules that make up living things. Carbohydrates include sugars and starches and are used for energy storage. Lipids are composed of fatty acids and glycerol and function in energy storage, protection, and insulation. Proteins contain amino acids and perform a variety of functions including growth, energy production, and pH buffering. Nucleic acids like DNA and RNA contain nucleotides and store and transmit genetic information that directs cellular functions. These macromolecules are formed through dehydration synthesis and broken down through hydrolysis.
Lipids are a group of naturally occurring molecules that include fats, waxes, sterols, and fat-soluble vitamins. They serve important functions like storing energy, signaling, and as structural components of cell membranes. The document defines lipids and discusses their chemistry, classifications, structures, and biological importance. Key points covered include that lipids are insoluble in water but soluble in organic solvents, and include triglycerides, fatty acids, and other compounds.
Eicosanoids are oxygenation products of arachidonic acid (AA), a polyunsaturated fatty acid found in animal and plant cell membranes. AA is released from membranes by phospholipase A2 and metabolized via either the cyclooxygenase pathway to form prostaglandins, prostacyclin, and thromboxanes, or the lipoxygenase pathway to form leukotrienes. These eicosanoids are involved in various physiological functions including inflammation, smooth muscle tone, blood coagulation, reproduction, and GI secretion. Clinical applications of prostaglandins include female and male reproductive health, inflammation and immunity, gastrointestinal, respiratory, musculoskeletal, cardiovascular, ocular health, and cancer.
Aerobic organisms continuously produce reactive free radicals through respiration, metabolism and phagocytosis. Approximately 1-2% of oxygen consumed is converted into superoxide radicals by the respiratory chain, one of the main sources of free radicals in cells. While oxygen is necessary for life, its partial reduction can produce reactive oxygen species (ROS) that damage living systems. The body has multiple antioxidant defenses to combat ROS, including superoxide dismutase, catalase, glutathione peroxidase and vitamin C, which help convert ROS into less reactive species and protect biomolecules from oxidative damage.
Lipids are a group of naturally occurring molecules that include fats, waxes, sterols, and fat-soluble vitamins. They serve important functions like energy storage, signaling, and as structural components of cell membranes. The main classes of lipids are neutral fats/triglycerides (consisting of glycerol and fatty acids), phospholipids, and sterols. Fatty acids can be saturated or unsaturated, and polyunsaturated fatty acids like omega-3 and omega-6 are essential nutrients. Lipids are insoluble in water but soluble in organic solvents, and are an important energy source in animals and plants.
This document discusses carcinogens and cancer. It defines cancer as abnormal cell growth that can invade other tissues and spread to other parts of the body. Carcinogens are substances that can cause cancer. Chemical carcinogens include aromatic hydrocarbons, aromatic amines, and chemicals containing epoxide, organohalogen, and nitroso groups. Carcinogens can damage DNA directly or require metabolic activation. Factors that influence cancer development include dose of exposure, lifestyle factors like smoking, and inherited conditions. Engineering controls, personal protective equipment, hygiene practices, and proper waste disposal can reduce exposure to carcinogens.
This document discusses the importance of breastfeeding for infant health and development. It reviews several studies that show breastfeeding reduces the risk of morbidity and mortality from various infectious diseases like diarrhea, otitis media, neonatal sepsis, and respiratory infections. However, in many societies false beliefs interfere with breastfeeding and infants are commonly given prelacteal feeds or mixed feeding instead of being exclusively breastfed. The purpose of the study described is to examine the patterns of infectious diseases in non-breastfed infants compared to breastfed infants admitted to the hospital.
This document describes a study comparing plasma osmolarity in healthy breastfed and non-breastfed infants. The study included breastfed and non-breastfed infants between 1-6 months of age. Blood samples were collected and analyzed for various biomarkers including glucose, BUN, sodium, potassium, total protein, and albumin levels. Plasma osmolarity was then directly estimated and calculated based on biomarker levels. Results showed breastfed infants had significantly lower glucose, BUN, and plasma osmolarity levels compared to non-breastfed infants. The study concluded breastfeeding provides a lower solute load compared to formula feeding, resulting in lower plasma osmolarity in healthy breastfed infants.
1. Metabolism refers to the chemical processes that take place in living organisms to sustain life. It includes breaking down nutrients into smaller units and building up complex molecules.
2. Glucose, fats, and proteins are broken down through various pathways to ultimately form acetyl CoA, which enters the citric acid cycle to generate energy in the form of ATP. Less oxygen results in lactic acid formation from glucose.
3. The electron transport chain uses oxygen to convert products of the citric acid cycle into large amounts of ATP, the main energy currency of cells. Fatty acids yield more ATP than glucose due to their carbon-hydrogen bonds.
The Tufts Robotics Club attended the Trinity College Fire Fighting Robot Contest where they won both the team and individual categories of the Olympiad tournament. This marked the club's fourth win since 2014. They competed in both the mechanical robot competition and trivia-based Olympiad. For the robot competition, Tufts engineered a robot that followed the right wall of the maze and used a fire extinguisher to put out a candle, navigating the maze faster than other teams. In the Olympiad, they demonstrated their strong theoretical engineering knowledge, with sophomore Faizan Muhammad winning individually. The success at this competition aims to attract new recruits to the club.
A glucose tolerance test (GTT) checks how the body metabolizes blood sugar levels over time. There are two main types: an oral GTT where glucose is ingested, and an intravenous GTT where glucose is injected. It is commonly used to screen for prediabetes and diabetes, especially in obese, pregnant, or high-risk individuals. The test involves fasting overnight, then drinking a glucose solution and having blood drawn over 3 hours to analyze the body's insulin response and how quickly glucose is cleared from the blood. Results are interpreted according to WHO criteria, with different glucose level thresholds indicating normal, prediabetes, or diabetes status.
This document summarizes a study that compares the performance of two state observers - a sliding mode observer with super-twisting algorithm (STSMO) and a high gain observer (HGO) - for estimating unmeasured states of a quadrotor UAV. The paper designs each observer and then applies a second order sliding mode control technique using the estimated states to control the quadrotor. Simulations show the performance of each observer under the same control scheme and perturbations. The study aims to compare the observers' characteristics for state estimation of the quadrotor system to determine the best observer for real-time applications given system uncertainties and noise.
This study examined the usage of social media by students at the FJMU Lahore. A questionnaire was distributed to 415 students to gather data on their social media usage patterns and behaviors. The results found that 95.9% of students used social media daily, with 77% spending 1-2 hours per day on social media. Major purposes of usage were communication (94%) and entertainment (93%), though 46% also used it for studying. Students reported social media could waste time and distract from studies, but could also be useful for sharing medical videos, knowledge and study guidelines. The study concludes social media distracts students and encourages more non-educational activities, so students should utilize it more for academic purposes and limit entertainment usage
Dr. Muhammad Mustansar has achieved international recognition for his work. He holds a world record and has had his research published in international posters and books. His accomplishments demonstrate expertise in his field at a global level.
This document discusses guidelines for drug use during lactation. It begins by outlining principles from the Quran and Hadith regarding breastfeeding. It then discusses anatomy related to lactation and hormonal regulation. Guidelines for chemotherapy during lactation emphasize using drugs with minimal infant exposure and avoiding unnecessary drugs. Several classes of drugs are discussed, identifying those that are generally safe to use during lactation like beta-blockers, thiazides, ACE inhibitors, warfarin, inhaled asthma medications, and corticosteroids. Drugs that should be avoided include phenobarbital, primodone, and clonazepam. The document also covers stimulants and inhibitors of lactation.
Cellular respiration is the process by which cells break down glucose and other organic molecules to obtain energy in the form of ATP. It occurs in three main stages: glycolysis, the citric acid cycle, and oxidative phosphorylation. During glycolysis, glucose is broken down to form pyruvate in the cytoplasm. In the citric acid cycle, pyruvate enters the mitochondria and is further oxidized, producing NADH, FADH2, and ATP. During oxidative phosphorylation, electrons from NADH and FADH2 are passed through an electron transport chain in the mitochondrial inner membrane. Their energy is used to pump protons out of the matrix, establishing a proton gradient. ATP synthase uses this gradient to
Wellstart International published international posters and books in San Diego, USA in 1993. The document references Wellstart International publishing international posters and books in 1993 in San Diego, USA.
Main Java[All of the Base Concepts}.docxadhitya5119
This is part 1 of my Java Learning Journey. This Contains Custom methods, classes, constructors, packages, multithreading , try- catch block, finally block and more.
Walmart Business+ and Spark Good for Nonprofits.pdfTechSoup
"Learn about all the ways Walmart supports nonprofit organizations.
You will hear from Liz Willett, the Head of Nonprofits, and hear about what Walmart is doing to help nonprofits, including Walmart Business and Spark Good. Walmart Business+ is a new offer for nonprofits that offers discounts and also streamlines nonprofits order and expense tracking, saving time and money.
The webinar may also give some examples on how nonprofits can best leverage Walmart Business+.
The event will cover the following::
Walmart Business + (https://business.walmart.com/plus) is a new shopping experience for nonprofits, schools, and local business customers that connects an exclusive online shopping experience to stores. Benefits include free delivery and shipping, a 'Spend Analytics” feature, special discounts, deals and tax-exempt shopping.
Special TechSoup offer for a free 180 days membership, and up to $150 in discounts on eligible orders.
Spark Good (walmart.com/sparkgood) is a charitable platform that enables nonprofits to receive donations directly from customers and associates.
Answers about how you can do more with Walmart!"
LAND USE LAND COVER AND NDVI OF MIRZAPUR DISTRICT, UPRAHUL
This Dissertation explores the particular circumstances of Mirzapur, a region located in the
core of India. Mirzapur, with its varied terrains and abundant biodiversity, offers an optimal
environment for investigating the changes in vegetation cover dynamics. Our study utilizes
advanced technologies such as GIS (Geographic Information Systems) and Remote sensing to
analyze the transformations that have taken place over the course of a decade.
The complex relationship between human activities and the environment has been the focus
of extensive research and worry. As the global community grapples with swift urbanization,
population expansion, and economic progress, the effects on natural ecosystems are becoming
more evident. A crucial element of this impact is the alteration of vegetation cover, which plays a
significant role in maintaining the ecological equilibrium of our planet.Land serves as the foundation for all human activities and provides the necessary materials for
these activities. As the most crucial natural resource, its utilization by humans results in different
'Land uses,' which are determined by both human activities and the physical characteristics of the
land.
The utilization of land is impacted by human needs and environmental factors. In countries
like India, rapid population growth and the emphasis on extensive resource exploitation can lead
to significant land degradation, adversely affecting the region's land cover.
Therefore, human intervention has significantly influenced land use patterns over many
centuries, evolving its structure over time and space. In the present era, these changes have
accelerated due to factors such as agriculture and urbanization. Information regarding land use and
cover is essential for various planning and management tasks related to the Earth's surface,
providing crucial environmental data for scientific, resource management, policy purposes, and
diverse human activities.
Accurate understanding of land use and cover is imperative for the development planning
of any area. Consequently, a wide range of professionals, including earth system scientists, land
and water managers, and urban planners, are interested in obtaining data on land use and cover
changes, conversion trends, and other related patterns. The spatial dimensions of land use and
cover support policymakers and scientists in making well-informed decisions, as alterations in
these patterns indicate shifts in economic and social conditions. Monitoring such changes with the
help of Advanced technologies like Remote Sensing and Geographic Information Systems is
crucial for coordinated efforts across different administrative levels. Advanced technologies like
Remote Sensing and Geographic Information Systems
9
Changes in vegetation cover refer to variations in the distribution, composition, and overall
structure of plant communities across different temporal and spatial scales. These changes can
occur natural.
How to Make a Field Mandatory in Odoo 17Celine George
In Odoo, making a field required can be done through both Python code and XML views. When you set the required attribute to True in Python code, it makes the field required across all views where it's used. Conversely, when you set the required attribute in XML views, it makes the field required only in the context of that particular view.
How to Manage Your Lost Opportunities in Odoo 17 CRMCeline George
Odoo 17 CRM allows us to track why we lose sales opportunities with "Lost Reasons." This helps analyze our sales process and identify areas for improvement. Here's how to configure lost reasons in Odoo 17 CRM
Executive Directors Chat Leveraging AI for Diversity, Equity, and InclusionTechSoup
Let’s explore the intersection of technology and equity in the final session of our DEI series. Discover how AI tools, like ChatGPT, can be used to support and enhance your nonprofit's DEI initiatives. Participants will gain insights into practical AI applications and get tips for leveraging technology to advance their DEI goals.
How to Setup Warehouse & Location in Odoo 17 InventoryCeline George
In this slide, we'll explore how to set up warehouses and locations in Odoo 17 Inventory. This will help us manage our stock effectively, track inventory levels, and streamline warehouse operations.
it describes the bony anatomy including the femoral head , acetabulum, labrum . also discusses the capsule , ligaments . muscle that act on the hip joint and the range of motion are outlined. factors affecting hip joint stability and weight transmission through the joint are summarized.
This slide is special for master students (MIBS & MIFB) in UUM. Also useful for readers who are interested in the topic of contemporary Islamic banking.
How to Fix the Import Error in the Odoo 17Celine George
An import error occurs when a program fails to import a module or library, disrupting its execution. In languages like Python, this issue arises when the specified module cannot be found or accessed, hindering the program's functionality. Resolving import errors is crucial for maintaining smooth software operation and uninterrupted development processes.
2. • STEPS
• Taking Paraffin Sections to Water
• Dehydration and Clearing of Sections in Xylene for
Mounting
• Blotting Sections Dry Before Mounting
• Mounting of Sections in DPX 3
• Ziehl-Neelsen Technique for Acid-Fast Bacilli
• Gram-Twort Modification for Bacteria in Paraffin
Sections
• Periodic Acid Schiff Technique
• Periodic Acid Schiff / Alcian Blue
• Buffered Congo Red Method for Amyloid
• Perls’ Prussian Blue Method for Haemosiderin
•
3. • TAKING PARAFFIN SECTIONS TO WATER
•
• SAFETY NOTE: Turn on the exhaust system
before commencing.
• Wear protective clothing, gloves and safety
glasses during the procedure.
•
4. • xylene 2 minutes
• xylene 2 minutes
• absolute alcohol 1 minute
• absolute alcohol 2 minutes
• 70% alcohol 30 seconds
• water
• Take the slides through the various solutions as follows:
• 1. Place slides in slide holder.
• 2. Lift lid off staining dish, and immerse the slides in the first solution with
agitation.
• 3. Replace lid and allow slides to remain in solution for the specified time
with periodic agitation.
• 4. Remove slides from the solution (with agitation) and tilt slide holder to
allow excess solution to drain
• before transferring it to the next solution.
• 5. Continue in this manner through the remaining solutions for the
specified times
• Times can be reduced if slides are agitated constantly
5. • DEHYDRATION AND CLEARING OF SECTIONS IN XYLENE
BEFORE MOUNTING
•
• SAFETY NOTE: Turn on the exhaust system before commencing.
• Wear protective clothing, gloves and safety glasses during the procedure.
• Dehydration 70% alcohol 15 seconds
• absolute alcohol 1 minute
• absolute alcohol 2 minutes
• Clearing xylene 15 seconds
• xylene 15 seconds
• Take the slides through the various solutions as follows:
• 1. Place slides in slide holder.
• 2. Lift lid off staining dish, and immerse the slides in the first solution with
agitation.
• 3. Replace lid and allow slides to remain in solution for the specified time with
periodic agitation.
• 4. Remove slides from the solution (with agitation) and tilt slide holder to allow
excess solution to drain
• before transferring it to the next solution.
• 5. Continue in this manner through the remaining solutions for the specified times.
• Times can be reduced if slides are agitated constantly.
•
6. BLOTTING DRY AND CLEARING OF
SECTIONS
SAFETY NOTE: Turn on the exhaust system before commencing.
Wear protective clothing, gloves and safety glasses during the
procedure.
Blotting Dry
1. Place slide face down carefully on blotting paper.
2. Fold blotting paper over slide over and apply gentle pressure
to dry slide.
3. Lift slide and move to dry section of blotting paper and
repeat until section is completely dry.
4. Allow section to air dry if necessary before clearing.
Clearing xylene 15 seconds
xylene 15 seconds
Clear dried section by dipping the slide in xylene for specified
times and mount in DPX.
7. MOUNTING OF SECTIONS
SAFETY NOTE: Turn on the exhaust system before
commencing.
Wear protective clothing, gloves and safety glasses during the
procedure.
NB If section is dry, dip in xylene before mounting the
coverslip
1. Remove slide from slide holder.
2. Carefully dry the BACK of the slide with a tissue.
3. Lay the slide on blotting paper or bench-coat on the fume
bench.
4. Place a drop of DPX mounting medium on the section.
5. Carefully place a cover-slip over the DPX ensuring that it
covers the section.
6. Remove any bubbles by jiggling the coverslip gently.
7. Place mounted slide on small cardboard slide holder and
place in 50oC oven for a few hours.
8. Allow mounted slide to dry lying flat for at least 1 week
8. ZIEHL-NEELSEN TECHNIQUE FOR ACID-FAST BACILLI
(ZN)
(Ziehl, 1882; Neelsen, 1883)
The histological method is similar to the classical bacteriological technique
that depends upon the
resistance of certain bacilli to decolourisation by acid alcohol after being
stained with hot carbol-fuchsin.
Fixation
Most fixatives can be used. Avoid Carnoy which removes lipid from the bacilli
which makes them less
acid-fast. Formalin, especially when prolonged, is said to reduce acid-fastness,
but specimens usually stain
perfectly satisfactorily. The treatment of formalin fixed sections with 0.5%
ammonium hydroxide before
staining may improve the brightness of color of acid-fast bacilli, but this is
seldom necessary and may
detach the sections from the slide.
Sections
Thin (3-5 μ) sections.
SAFETY NOTE: Turn on the exhaust system before staining.
Wear protective clothing, gloves and safety glasses during the staining
procedure.
9. • ZN Staining Procedure
• ZN Procedure - Slide Method
• 1. Place a rectangle of filter paper over the section (to prevent precipitation of the stain) and
flood the
• slide with carbol-fuchsin.
• 2. Warm the slide until the stain begins to steam; this can be conveniently done by the flame
from a
• throat swab soaked in alcohol.
• 3. Leave for 5 minutes.
• 4. Wash in tap water for 2 minutes.
• 5. Differentiate in acid alcohol (3% HCl in 95% ethanol) until no more color runs from the
slide.
• 6. Rinse in water to remove acid alcohol.
• 7. Counterstain in acidified methylene blue for 30 seconds.
• 8. Wash in water, dehydrate, clear and mount in synthetic resin eg DPX.
• OR
• ZN Procedure - Coplin Jar Method
• 1. Take sections to water.
• 2. Place the working solution in a coplin jar and pre-heat in 58 - 60oC waterbath for 10 mins
• 3. Place the slide in the warmed vessel of carbol-fuchsin for at least 30 minutes at 58 - 60oC.
• 4. Remove slide from coplin jar and wash in tap water for 2 minutes.
• 5. Differentiate in acid alcohol (3% HCl in 95% ethanol) until no more color runs from the
slide.
• 6. Rinse in water to remove acid alcohol.
• 7. Counterstain in acidified methylene blue for 30 seconds.
• 8. Wash in water, dehydrate, clear and mount in synthetic resin eg DPX.
10. • Results
• Acid-fast bacilli: red
• Other bacteria: blue
• Cells and their nuclei: blue
• Red blood cells should retain a slight red color.
• Notes
• 1 Since this method involves the use of both acid and alcohol decolourisation, the risk of
mistaking the
• non-pathogenic acid-fast bacilli for the tubercle bacillus is decreased. In sections the tubercle
bacilli
• will be found in the abnormal areas (tubercles) and this risk is slight. The non-pathogenic
acid-fast
• bacilli that are found in butter, milk, or cerumen are seldom acid-fast, but the smegma
bacillus may
• require prolonged alcohol decolourisation.
• 2 Counterstaining should be light, especially in sections containing nuclear material, such as
lymphoid
• tissue. Heavy counterstaining makes the identification of acid-fast bacilli difficult and may
color
• them purple.
• 3 Basic fuchsin specified for Schiff’s reagent may not give satisfactory results.
• 4 Control sections of known positive tuberculous material with abundant acid-fast bacilli are
valuable
• in that they indicate that the staining technique is satisfactory when several negative sections
are
• being examined. They also give an idea of the color of the acid-fast bacilli; this may vary from
a
• light to a dark red.
11. • The leprosy bacillus is more easily decolourised than the
tubercle bacillus, and differentiation must be
• carefully controlled. A faint residual red color in the tissues is
especially important, and sections that do
• not show this may be unreliable for the exclusion of leprosy.
The Fite-Faraco modification (Faraco, 1938;
• Fite, Cambre and Turner, 1947) for leprosy bacilli is similar to
the standard Ziehl-Neelsen technique, but
• the paraffin wax is removed from the sections with two
changes of one part of groundnut oil, cottonseed
• oil or olive oil and two parts xylene for 10 minutes each.
Wade (1952) used two parts of rectified
• turpentine and one part of liquid petrolatum for the same
purpose. After either of these variants the
• sections are drained, blotted until opaque and placed directly
in water; the residual oil in the sections helpsprevent
shrinkage.
•
12. • Reagent Preparation
• 1. Carbol Fuchsin
• Basic Fuchsin 1.0 g
• Absolute Ethanol 10 mL
• 5% phenol in distilled water 100 mL
• Dissolve the basic fuchsin in the alcohol, then mix with
the phenol solution. Filter.
• 2. Acid Alcohol (3% HCl)
• 3% hydrochloric acid in 95% alcohol.
• 3. Acidified Methylene Blue Counterstain
• Methylene Blue 0.25 g
• Glacial Acetic Acid 1 mL
• Distilled Water 99 mL
13. • THE GRAM-TWORT MODIFICATION FOR BACTERIA
IN PARAFFIN SECTIONS
•
• The following modification of the Twort (1924) method for bacteria
has the advantages of easier
• differentiation and a better color contrast compared with the other
Gram techniques (Ollett, 1947, 1951).
• The sections are easy to examine for long periods without eye
strain.
• Fixation
• Formalin; other fixative can be used.
• Sections
• Thin (3-5 μm) paraffin sections.
• Safety Note: Turn on the exhaust system before staining.
• Wear protective clothing, gloves and safety glasses during the
staining procedure.
14. Sections
Thin (3-5 μm) paraffin sections.
Safety Note: Turn on the exhaust system before staining.
Wear protective clothing, gloves and safety glasses during the staining
procedure.
Procedure
1 Stain in 1% crystal violet for 3-4 minutes.
2 Wash quickly in distilled water.
3 Treat with Gram’s iodine for 3 minutes.
4 Wash quickly in distilled water and blot dry.
5 Decolourise briefly with 2% acetic acid in absolute alcohol until no
more color comes away - the
section should be a dirty straw color at this stage.
6 Wash quickly in distilled water.
7 Counterstain in Modified Twort Stain in closed coplin jar for 5
minutes.
8 Wash quickly in distilled water.
9 Decolourise quickly and carefully in 2% acetic acid in absolute alcohol
until no more red color
comes away (a few seconds).
10 Clear in xylene and mount in a synthetic resin medium eg DPX
15. Results Gram positive bacteria: blue-black
Gram negative bacteria: pink
Nuclei: red
Cytoplasm: light green
Red blood cells: green
Principle of Gram Stain
Stain Gram-Pos Bacteria Gram-Neg Bacteria
Crystal Violet with Iodine Mordant Blue-Black Black
Acid Alcohol Blue-Black Colourless
Methyl Red Blue-Black Pink
If sections are exposed too long to alcohol, the Gram-positive bacteria will
also decolourise.
Most bacteria, especially in large numbers stain a pale grey color with
haematoxylin.
With all stains for microbes, it is essential that known positive control slides
are stained along with the section.
16. Reagents
1. Crystal Violet
Crystal violet (CI 42555 ) 2.0 g; 95% alcohol 20.0 ml; ammonium oxalate 0.8 g;
distilled water 80.0
mL. Dissolve dye in the alcohol & the ammonium oxalate in the dH2O, mix
together. Mixture stable
2-3 years.
2. Grams Iodine
1.0 g Iodine crystals (harmful); 2.0 g potassium iodide (harmful); 300.0 mL distilled
water
Dissolve KI in 2-3 mL only dH2O - the crystals will dissolve and the solution will
become very cold.
Dissolve the iodine crystals in the conc KI soln. Dilute mixture with the remainder
of the dH2O
3. Modified Twort’s Stain - Stock Solution (stable 1 year)
0.2% Neutral Red in 95% Ethanol 90 mL
0.2% Fast Green FCF in 95% Ethanol 10 mL
4. Modified Twort’s Stain - Working Solution (prepare fresh)
Dilute 1 volume of the stock solution with 3 volumes of distilled water.
5. Acid Alcohol (2% Acetic)
2% acetic acid in absolute alcohol.
17. • PERIODIC ACID SCHIFF TECHNIQUE (PAS)
• Sections are oxidised by the periodic acid resulting in the
formation of aldehyde groups. These then react
• with Schiff’s reagent (a leucofuchsin) to restore the quinoid
chromophoric grouping, giving a magenta
• coloured final product to the PAS positive substances.
• Procedure
• 1 Take sections to water
• 2 Remove mercuric deposit (if present) with iodine
thiosulphate
• 3 Oxidize with Periodic Acid 5 mins
• 4 Wash well in running water for 5 minutes
• 5 Rinse with distilled water
• 6 Stain with Schiff's Reagent 15-20 mins
18. • 6 Stain with Schiff's Reagent 15-20 mins
• 7 Wash well in running tap water 5 mins
• 8 Stain nuclei with Harris Haematoxylin 1 min
• 9 Wash in running tap water 2 mins
• 10 Differentiate briefly (1-2 seconds) with acid alcohol
• 11 Wash and blue nuclei in ammonia water
• 12 Wash briefly in running tap water
• 13 Blue in ammonia water and running tap water
• 14 Dehydrate quickly in alcohol, clear in xylene and mount in DPX
• Technical Points
• 1. (step 6) - Schiff's reagent deteriorates rapidly if not kept in a closed container. When
a pinkish
• discolouration appears, discard the reagent.
• 2. (step 7) - Washing not only removes any excess reagent from the section, but also
promotes the
• development of the rich magenta color. Too gentle washing will result in a strong
artefactual red
• stained background due to the action of the powerful dye basic fuchsin, formed from
the
• destabilisation of the leuco fuchsin by loss of sulphurous acid to the watery
environment.
• 3. (step 10) - Over differentiation can lead to the eventual decolourisation of PAS
positive material.
•
19. • Results
• Simple polysaccharides, neutral mucosubstances,
some macro mucosubstances and basement
membranes
• are PAS positive (Magenta in color)
•
• Reagents
• 1. 1% Aqueous Periodic Acid
• 2. Schiff’s Reagent
• 3. Harris’s Haematoxylin
• 4. Ammonia Water
• 5. Acid Alcohol - 1% HCl in 70% ethanol
•
20. • PERIODIC ACID SCHIFF/ALCIAN BLUE TECHNIQUE (PAS/AB)
•
• Sections are oxidised by the periodic acid resulting in the
formation of aldehyde groups. These then react with
Schiff’s reagent (a leucofuchsin) to restore the quinoid
chromophoric grouping, giving a magenta coloured final
product to the PAS positive substances.
• By first treating the section with Alcian Blue the acid
mucins will stain and therefore will not react when
• the section is subsequently stained with the PAS method.
The PAS will stain neutral mucins and
• carbohydrates, red.
• Alcian Blue/PAS Staining Procedure
• 1. Take sections to water.
• 2. Stain in 1% Alcian Blue solution for 10-15 minutes.
• 3. Wash in running water for 2 minutes.
21. • 4. Rinse in distilled water.
• 5. Oxidize with Periodic Acid 5 mins
• 6. Wash in running water for 5 minutes
• 7. Rinse with distilled water
• 8. Stain with Schiff's Reagent 15-20 mins
• 9. Wash well in running tap water 5 mins
• 10. Stain nuclei with Harris Haematoxylin 1 min
• 11. Wash in running tap water 2 mins
• 12. Differentiate briefly (1-2 seconds) with acid alcohol
• 13. Wash briefly in running tap water
• 14. Blue in ammonia water and running tap water
• 15. Dehydrate quickly in alcohol, clear in xylene and
mount in DPX
22. • Technical Points
• 4. (step 6) - Schiff's reagent deteriorates rapidly if not
kept in a closed container. When a pinkish
• discolouration appears, discard the reagent.
• 5. (step 7) - Washing not only removes any excess
reagent from the section, but also promotes the
• development of the rich magenta color. Too gentle
washing will result in a strong artefactual red
• stained background due to the action of the powerful
dye basic fuchsin, formed from the
• destabilisation of the leuco fuchsin by loss of
sulphurous acid to the watery environment.
• 6. (step 12) - Over differentiation can lead to the
eventual decolourisation of PAS positive material.
•
23. • Results
• Neutral mucins magenta
• Acid mucins blue
• Mixtures of above blue/purple
• Nuclei deep blue
• Basement membranes magenta
• Reagents
• 1 1% Alcian Blue in 3% Acetic Acid (pH 2.5)
• 2 0.5 % Aqueous Periodic Acid
• 3 Schiff’s Reagent
• 4. Harris’s Haematoxylin
• 5. Ammonia Water
• 6. Acid Alcohol - 1% HCl in 70% ethanol
•
24. • BUFFERED CONGO RED METHOD FOR AMYLOID (EASTWOOD AND
COLE 1971)
• Fixative
• Buffered formal-saline
• Safety Note: Turn on the exhaust system before staining.
• Wear protective clothing, gloves and safety glasses during the
staining procedure.
• Procedure
• 1. Take sections to water.
• 2. Stain in Harris’s Haematoxylin for 30 seconds.
• 3. Rinse in tap water.
• 4. Differentiate in acid alcohol for a few seconds (2 - 3dips)
• 5. Rinse in tap water.
• 6. Blue in ammonia water followed by running tap water.
• 7. Stain in 0.5% Congo Red for 10-20 minutes.
• 8. Differentiate in 70% alcohol for a few seconds (2 - 3 dips).
• 9. Blot dry.
• 10. Clear in xylene and mount in DPX.
25. • Results
• Amyloid orange to red
• Elastic tissue, eosinophils orange to red
• Nuclei: blue
• Notes
• 1. Dichroism is pronounced and can assist in distinguishing amyloid from other tissue
components.
• 2. The stain has a permanent shelf life but may require occasional filtering.
• Reagents
• 1. pH 10.0 Sorensen-Walbum buffer
• 0.1M Glycine (MW 72.07) 30 mL
• 0.1M Sodium Chloride (MW 58.5) 30 mL
• 0.1M Sodium Hydroxide (MW40.0) 40 mL
• Mix together and check pH.
• 2. 0.5% Congo Red Stain
• Congo Red 0.5g
• Absolute alcohol 50ml
• pH 10.0 Sorensen-Walbum Buffer 50ml
• 3. Harris’s Haematoxylin
• 4. Ammonia Water
• 5. Acid Alcohol - 1% HCl in 70% ethanol
• 6. 70% Alcohol - 70% ethanol
•
26. • PERLS’ PRUSSIAN BLUE METHOD FOR HAEMOSIDERIN
(Perls)
•
• Ferric iron combines with potassium ferrocyanide to form
the insoluble Prussian blue precipitate as
• Follows:
• FeCl3 + K4Fe(CN)6 = KFeFe(CN)6¯ + 3KCl
• Fixation
• Neutral formalin (acid fixatives and potassium dichromate
should be avoided).
• Sections
• Thin (3-5 μ) paraffin sections.
• SAFETY NOTE: Turn on the exhaust system before staining.
• Wear protective clothing, gloves and safety glasses during
the staining procedure
27. Procedure
1. Take sections to water
2. Rinse well in distilled water.
3. Transfer sections to a mixture of equal parts of 2%
Potassium Ferrocyanide and 2% Hydrochloric
Acid for 20-30 minutes.
4. Wash in tap water and then rinse in distilled water.
5. Counterstain in filtered 1% neutral red for 1 minute.
6. Rinse in tap water.
7. Rapidly dehydrate in absolute alcohol, clear and mount.
Results
Haemosiderin and ferric salts: deep blue
Tissues and nuclei: red
Cytoplasm pink
Erythrocytes yellow
28. Notes
1 More pronounced staining is obtained by heating the ferrocyanide to 37oC.
2 If the stain fades it may be revived by treating with 10 vol H2O2.
3 Tap water must be avoided at all times. The distilled water must be iron-
free, and the hydrochloric
acid must be of analytical grade, or it will contain iron.
Reagents
1. 2% Aqueous Solution of Hydrochloric Acid
2. 2% Aqueous Solution of Potassium Ferrocyanide
3. Perls Working Solution:
Mix equal parts of 2% hydrochloric acid and 2% potassium ferrocyanide
solution JUST before
use.
4. 1% Neutral Red
Neutral red (CI 50040) 1.0 g
Distilled water 99.0 mL
Glacial acetic acid 1.0 mL