This document describes the process and principles of immunofluorescence assays. It discusses both direct and indirect immunofluorescence techniques. Direct immunofluorescence involves applying fluorescently-labeled antibodies directly to tissue samples to detect antigens. Indirect immunofluorescence uses unlabeled patient antibodies detected using secondary fluorescently-labeled anti-human antibodies to identify antibodies in serum. The document provides detailed steps for performing indirect immunofluorescence assays including preparing antigen slides, making serum dilutions, incubation, washing, and examination under a fluorescence microscope.
This document provides instructions for using an enzyme-linked immunosorbent assay (ELISA) kit to detect the mycotoxin deoxynivalenol (DON) in cereal grains and animal feeds. The kit uses a competitive ELISA format where DON in samples competes with DON conjugated to an enzyme for binding to an anti-DON antibody. The amount of enzyme bound is then measured to determine the DON concentration in samples, which can range from 0.5 to 10 parts per million after accounting for sample dilutions. Proper sample extraction in water and buffer is required before performing the assay according to the provided multi-step protocol.
This document outlines procedures for performing microbial limit tests on pharmaceutical products. The tests are designed to qualitatively or quantitatively estimate the number of viable aerobic microorganisms present or detect designated microbial species. Several methods are described, including membrane filtration, pour plate, spread plate, and multiple tube dilution. Specific procedures are provided for testing for total aerobic count, E. coli, and Salmonella. Controls and interpretation of results are also described to validate the testing methods.
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.
The document describes four main types of ELISA assays: direct, indirect, sandwich, and competitive. It provides details on the procedure for each type. Direct ELISA uses one antibody directly conjugated to an enzyme for detection. Indirect ELISA uses an unlabeled primary antibody detected by a labeled secondary antibody, allowing for signal amplification. Sandwich ELISA requires two antibodies that bind to different epitopes of the target antigen. Competitive ELISA measures competition between a labeled antigen and unlabeled antigen in a sample.
The complete guide to antibody detection by immunofluorescence techniqueCandy Swift
The document provides instructions for using immunofluorescence techniques to detect antibodies. It describes how to prepare cell samples and reagents, and outlines two main methods - indirect immunofluorescence and cell membrane fluorescence staining. Indirect immunofluorescence involves incubating cell samples with antibody samples to be tested, then a fluorescent secondary antibody, and viewing under a microscope. Cell membrane fluorescence staining uses living cell suspensions incubated at 4°C with antibody samples and fluorescent antibodies to stain just the cell membrane.
This document provides information about an enzyme-linked immunosorbent assay (ELISA) for quantitatively detecting levels of aflatoxin M1 in urine. Aflatoxin M1 is a metabolite of the toxic and carcinogenic aflatoxin B1 fungus and can be detected in urine after ingestion of aflatoxin B1. The ELISA uses antibodies coated on microwells to capture aflatoxin M1 in urine samples, and a detection system to quantify levels. Validation studies found the assay accurately recovered spiked levels of aflatoxin M1 in urine samples and could help monitor populations at risk of aflatoxin exposure.
The document outlines various methods used to test the efficacy of disinfectants, including carrier tests, suspension tests, and practical tests. Carrier tests involve contaminating a thread with bacteria and exposing it to disinfectants. Suspension tests measure a disinfectant's ability to kill bacteria suspended in its solution. Practical tests evaluate disinfectants under real-world conditions. The document also describes the phenol coefficient test, which compares a disinfectant's effectiveness to that of phenol, and the filter paper test, which detects zones of bacterial inhibition around treated disks.
This document provides instructions for using an enzyme-linked immunosorbent assay (ELISA) kit to detect the mycotoxin deoxynivalenol (DON) in cereal grains and animal feeds. The kit uses a competitive ELISA format where DON in samples competes with DON conjugated to an enzyme for binding to an anti-DON antibody. The amount of enzyme bound is then measured to determine the DON concentration in samples, which can range from 0.5 to 10 parts per million after accounting for sample dilutions. Proper sample extraction in water and buffer is required before performing the assay according to the provided multi-step protocol.
This document outlines procedures for performing microbial limit tests on pharmaceutical products. The tests are designed to qualitatively or quantitatively estimate the number of viable aerobic microorganisms present or detect designated microbial species. Several methods are described, including membrane filtration, pour plate, spread plate, and multiple tube dilution. Specific procedures are provided for testing for total aerobic count, E. coli, and Salmonella. Controls and interpretation of results are also described to validate the testing methods.
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.
The document describes four main types of ELISA assays: direct, indirect, sandwich, and competitive. It provides details on the procedure for each type. Direct ELISA uses one antibody directly conjugated to an enzyme for detection. Indirect ELISA uses an unlabeled primary antibody detected by a labeled secondary antibody, allowing for signal amplification. Sandwich ELISA requires two antibodies that bind to different epitopes of the target antigen. Competitive ELISA measures competition between a labeled antigen and unlabeled antigen in a sample.
The complete guide to antibody detection by immunofluorescence techniqueCandy Swift
The document provides instructions for using immunofluorescence techniques to detect antibodies. It describes how to prepare cell samples and reagents, and outlines two main methods - indirect immunofluorescence and cell membrane fluorescence staining. Indirect immunofluorescence involves incubating cell samples with antibody samples to be tested, then a fluorescent secondary antibody, and viewing under a microscope. Cell membrane fluorescence staining uses living cell suspensions incubated at 4°C with antibody samples and fluorescent antibodies to stain just the cell membrane.
This document provides information about an enzyme-linked immunosorbent assay (ELISA) for quantitatively detecting levels of aflatoxin M1 in urine. Aflatoxin M1 is a metabolite of the toxic and carcinogenic aflatoxin B1 fungus and can be detected in urine after ingestion of aflatoxin B1. The ELISA uses antibodies coated on microwells to capture aflatoxin M1 in urine samples, and a detection system to quantify levels. Validation studies found the assay accurately recovered spiked levels of aflatoxin M1 in urine samples and could help monitor populations at risk of aflatoxin exposure.
The document outlines various methods used to test the efficacy of disinfectants, including carrier tests, suspension tests, and practical tests. Carrier tests involve contaminating a thread with bacteria and exposing it to disinfectants. Suspension tests measure a disinfectant's ability to kill bacteria suspended in its solution. Practical tests evaluate disinfectants under real-world conditions. The document also describes the phenol coefficient test, which compares a disinfectant's effectiveness to that of phenol, and the filter paper test, which detects zones of bacterial inhibition around treated disks.
This document provides information on testing the total aerobic microbial count in pharmaceutical products and materials. It defines the objective as estimating the number of viable aerobic organisms present to determine compliance. The methods section describes preparing samples by dissolving, diluting, grinding or emulsifying them, then plating serial dilutions and counting colonies to find the concentration of microorganisms. Precautions are outlined to avoid contamination during testing.
The document describes a study to detect Puccinia horiana, the causal agent of Chrysanthemum white rust disease, in planta using in situ hybridization. Key findings include:
1. Optimization of the in situ hybridization protocol included determining optimal proteinase K digestion time, formamide concentration during hybridization, and post-hybridization wash temperature for specific detection of P. horiana in chrysanthemum leaf tissue sections.
2. The optimized protocol successfully detected P. horiana infection with intense signal and high specificity, demonstrating an improvement over previous detection methods.
3. Future work will involve applying the optimized protocol to detect P. horiana infection in other plant tissues and samples from
The document discusses pyrogen testing techniques including the rabbit test and LAL (Limulus Amebocyte Lysate) test. It provides details on how to conduct the rabbit test, including temperature monitoring and criteria for a passing result. For the LAL test, it describes the mechanism, different methods (gel clot, turbidimetric, chromogenic), and procedures for confirming lysate sensitivity and determining endotoxin levels in samples. It notes that various pharmacopeias like IP, BP, and USP specify methods for the LAL test.
The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.
This document provides procedures for conducting a Microbial Limit Test (MLT). The test involves several steps: sample pretreatment, total aerobic count using membrane filtration or plate count methods, and examination for specified microorganisms like E. coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. Positive and negative controls are run alongside each test. The procedures describe preparing bacterial and fungal suspensions, inoculating various media, and incubating and examining plates to identify microbial growth or absence. Safety precautions like using clean gloves and running tests under laminar airflow are also outlined.
This document discusses procedures for evaluating parenteral products, including sterility testing, clarity testing, leakage testing, and potency testing. It focuses on sterility testing methods such as membrane filtration and direct inoculation. Key steps include incubating samples in culture media to check for microbial growth, which would indicate a failed sterility test. The document also covers pyrogen (fever-causing substance) testing using rabbits, where an intravenous injection is given and temperature changes are monitored; a large temperature increase would mean a failed pyrogen test. Sample sizes, incubation times, interpretation of results, and pass/fail criteria are outlined for these evaluation methods.
This document discusses antibiotic sensitivity testing, which determines the effectiveness of antibiotics against bacteria. It describes the purpose of testing as guiding treatment selection and monitoring resistance trends. Common testing methods are discussed, including disk diffusion, broth dilution, and agar dilution for minimum inhibitory concentration determination. The disk diffusion method is described in detail, outlining inoculum preparation, disk application, incubation, and result interpretation. Factors influencing testing and quality control are also covered.
This document discusses various staining techniques used to visualize bacteria under a microscope. It covers simple staining techniques like Gram staining and acid-fast staining, as well as methods to identify specific structures like volutin granules and bacterial spores. Gram staining uses dyes to differentiate between Gram-positive and Gram-negative bacteria based on their cell wall composition. Acid-fast staining targets bacteria with thick lipid cell walls like Mycobacterium tuberculosis. Specialized techniques employ unique dyes and fixation steps to highlight intracellular inclusions and endospores. Proper staining is crucial for bacterial identification and clinical diagnosis.
This document discusses antibiotic sensitivity testing (AST), which determines how effective antibiotics are against bacteria in vitro. AST is important for selecting the best antibiotic treatment for patients, monitoring antibiotic resistance trends, and accumulating epidemiological data. The Kirby-Bauer disk diffusion method is described, which uses antibiotic-impregnated disks placed on agar plates inoculated with bacteria. The diameter of inhibition zones around the disks after incubation indicates antibiotic sensitivity. Interpretive criteria classify results as sensitive, intermediate, or resistant. AST provides guidance for clinicians in choosing effective antibiotic therapy.
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 several staining techniques used to identify different characteristics of bacteria under a microscope. The Gram stain distinguishes between Gram-positive and Gram-negative bacteria and was an important early technique. The acid-fast stain identifies bacteria with waxy cell walls like Mycobacterium tuberculosis. The endospore stain reveals if a bacteria can form dormant endospores. Capsular staining highlights the capsules of virulent bacteria that are difficult to see with regular stains. Each technique has a specific multi-step procedure to prepare and differentially stain samples for examination.
Sterility test and modern microbiological methodsMohammed Fawzy
This document provides an overview of sterility testing and rapid microbiological methods. It discusses sterility testing, including definitions, common media used, methods for preparing different types of test products, incubation periods, growth promotion tests, and interpreting results. It also briefly introduces some rapid microbiological methods like ATP bioluminescence, colorimetric growth detection, and cytometry systems. The key purpose of sterility testing is to detect any viable microorganisms in pharmaceutical products or medical devices labeled as sterile.
This document discusses pyrogens, which are fever-producing substances that are byproducts of microorganisms like bacteria, molds, and viruses. It describes two main tests used to detect pyrogens - the rabbit test, which measures fever response in rabbits, and the Limulus amebocyte lysate (LAL) test, which detects pyrogens using a clotting reaction. The rabbit test involves injecting a sample into rabbits and monitoring their temperatures, while the LAL test uses a blood extract from horseshoe crabs that clots in response to pyrogens. Both tests are used to ensure parenteral solutions are free from pyrogens before use in humans.
Medical Microbiology Laboratory (biochemical tests - i)Hussein Al-tameemi
The document provides information on various biochemical tests used to identify bacteria, including enzymatic tests like catalase, coagulase, oxidase, and urease. It describes the basic principles, procedures, reagents, and results for each test. The catalase test detects the presence of the catalase enzyme, while the coagulase test detects coagulase production in Staphylococcus aureus. Positive and negative results are indicated by bubble or clot formation, respectively.
The document outlines various methods for microbial limit testing of pharmaceutical products and raw materials, including total aerobic microbial count testing using membrane filtration, plate count, and serial dilution methods. It discusses sample preparation, media types, sampling precautions, and defines terms like culture, cfu, and selective media. The goal is to estimate microbes present and determine if products meet BP, USP, or IP requirements.
This document describes bacterial staining techniques including Gram staining and acid-fast staining. Gram staining differentiates bacteria based on cell wall structure and stains Gram-positive bacteria purple and Gram-negative bacteria pink or red. Acid-fast staining identifies acid-fast bacteria that retain the primary stain carbolfuchsin despite decolorization. Procedures for both Gram staining and acid-fast staining of mixed bacterial samples are provided along with questions about the techniques.
The document discusses various evaluation tests performed on parenteral products, including sterility testing, clarity testing, leakage testing, pyrogen testing, and assay. Sterility testing involves incubating samples in culture media to check for microbial growth. Clarity testing examines products for visible particles. Leakage testing checks for cracks in ampoules. Pyrogen testing involves injecting products into rabbits to monitor for fever responses. Assay is performed to quantify the active ingredient in the parenteral preparation according to pharmacopeia methods. Proper testing helps ensure parenteral products are free of contaminants and contain the correct amount of active pharmaceutical ingredient.
This document provides instructions for using an ELISA kit to detect the mycotoxin zearalenone in cereal crops and animal feeds. It begins with an introduction to zearalenone and its health effects. It then describes the intended use, principle, reagents, materials, precautions, extraction procedure, and assay procedure for the zearalenone ELISA kit. The kit is designed to quantitatively detect zearalenone in samples through a competitive enzyme immunoassay.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
The document summarizes various physical and microbiological methods for testing semisolid dosage forms like ointments. It describes tests to evaluate rate of absorption, non-irritancy, rate of penetration, rate of drug release, viscosity, content uniformity, microbial content, and preservative efficacy. It also provides details on procedures for sterility testing using membrane filtration or direct inoculation methods.
This document provides information on testing the total aerobic microbial count in pharmaceutical products and materials. It defines the objective as estimating the number of viable aerobic organisms present to determine compliance. The methods section describes preparing samples by dissolving, diluting, grinding or emulsifying them, then plating serial dilutions and counting colonies to find the concentration of microorganisms. Precautions are outlined to avoid contamination during testing.
The document describes a study to detect Puccinia horiana, the causal agent of Chrysanthemum white rust disease, in planta using in situ hybridization. Key findings include:
1. Optimization of the in situ hybridization protocol included determining optimal proteinase K digestion time, formamide concentration during hybridization, and post-hybridization wash temperature for specific detection of P. horiana in chrysanthemum leaf tissue sections.
2. The optimized protocol successfully detected P. horiana infection with intense signal and high specificity, demonstrating an improvement over previous detection methods.
3. Future work will involve applying the optimized protocol to detect P. horiana infection in other plant tissues and samples from
The document discusses pyrogen testing techniques including the rabbit test and LAL (Limulus Amebocyte Lysate) test. It provides details on how to conduct the rabbit test, including temperature monitoring and criteria for a passing result. For the LAL test, it describes the mechanism, different methods (gel clot, turbidimetric, chromogenic), and procedures for confirming lysate sensitivity and determining endotoxin levels in samples. It notes that various pharmacopeias like IP, BP, and USP specify methods for the LAL test.
The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.
This document provides procedures for conducting a Microbial Limit Test (MLT). The test involves several steps: sample pretreatment, total aerobic count using membrane filtration or plate count methods, and examination for specified microorganisms like E. coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. Positive and negative controls are run alongside each test. The procedures describe preparing bacterial and fungal suspensions, inoculating various media, and incubating and examining plates to identify microbial growth or absence. Safety precautions like using clean gloves and running tests under laminar airflow are also outlined.
This document discusses procedures for evaluating parenteral products, including sterility testing, clarity testing, leakage testing, and potency testing. It focuses on sterility testing methods such as membrane filtration and direct inoculation. Key steps include incubating samples in culture media to check for microbial growth, which would indicate a failed sterility test. The document also covers pyrogen (fever-causing substance) testing using rabbits, where an intravenous injection is given and temperature changes are monitored; a large temperature increase would mean a failed pyrogen test. Sample sizes, incubation times, interpretation of results, and pass/fail criteria are outlined for these evaluation methods.
This document discusses antibiotic sensitivity testing, which determines the effectiveness of antibiotics against bacteria. It describes the purpose of testing as guiding treatment selection and monitoring resistance trends. Common testing methods are discussed, including disk diffusion, broth dilution, and agar dilution for minimum inhibitory concentration determination. The disk diffusion method is described in detail, outlining inoculum preparation, disk application, incubation, and result interpretation. Factors influencing testing and quality control are also covered.
This document discusses various staining techniques used to visualize bacteria under a microscope. It covers simple staining techniques like Gram staining and acid-fast staining, as well as methods to identify specific structures like volutin granules and bacterial spores. Gram staining uses dyes to differentiate between Gram-positive and Gram-negative bacteria based on their cell wall composition. Acid-fast staining targets bacteria with thick lipid cell walls like Mycobacterium tuberculosis. Specialized techniques employ unique dyes and fixation steps to highlight intracellular inclusions and endospores. Proper staining is crucial for bacterial identification and clinical diagnosis.
This document discusses antibiotic sensitivity testing (AST), which determines how effective antibiotics are against bacteria in vitro. AST is important for selecting the best antibiotic treatment for patients, monitoring antibiotic resistance trends, and accumulating epidemiological data. The Kirby-Bauer disk diffusion method is described, which uses antibiotic-impregnated disks placed on agar plates inoculated with bacteria. The diameter of inhibition zones around the disks after incubation indicates antibiotic sensitivity. Interpretive criteria classify results as sensitive, intermediate, or resistant. AST provides guidance for clinicians in choosing effective antibiotic therapy.
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 several staining techniques used to identify different characteristics of bacteria under a microscope. The Gram stain distinguishes between Gram-positive and Gram-negative bacteria and was an important early technique. The acid-fast stain identifies bacteria with waxy cell walls like Mycobacterium tuberculosis. The endospore stain reveals if a bacteria can form dormant endospores. Capsular staining highlights the capsules of virulent bacteria that are difficult to see with regular stains. Each technique has a specific multi-step procedure to prepare and differentially stain samples for examination.
Sterility test and modern microbiological methodsMohammed Fawzy
This document provides an overview of sterility testing and rapid microbiological methods. It discusses sterility testing, including definitions, common media used, methods for preparing different types of test products, incubation periods, growth promotion tests, and interpreting results. It also briefly introduces some rapid microbiological methods like ATP bioluminescence, colorimetric growth detection, and cytometry systems. The key purpose of sterility testing is to detect any viable microorganisms in pharmaceutical products or medical devices labeled as sterile.
This document discusses pyrogens, which are fever-producing substances that are byproducts of microorganisms like bacteria, molds, and viruses. It describes two main tests used to detect pyrogens - the rabbit test, which measures fever response in rabbits, and the Limulus amebocyte lysate (LAL) test, which detects pyrogens using a clotting reaction. The rabbit test involves injecting a sample into rabbits and monitoring their temperatures, while the LAL test uses a blood extract from horseshoe crabs that clots in response to pyrogens. Both tests are used to ensure parenteral solutions are free from pyrogens before use in humans.
Medical Microbiology Laboratory (biochemical tests - i)Hussein Al-tameemi
The document provides information on various biochemical tests used to identify bacteria, including enzymatic tests like catalase, coagulase, oxidase, and urease. It describes the basic principles, procedures, reagents, and results for each test. The catalase test detects the presence of the catalase enzyme, while the coagulase test detects coagulase production in Staphylococcus aureus. Positive and negative results are indicated by bubble or clot formation, respectively.
The document outlines various methods for microbial limit testing of pharmaceutical products and raw materials, including total aerobic microbial count testing using membrane filtration, plate count, and serial dilution methods. It discusses sample preparation, media types, sampling precautions, and defines terms like culture, cfu, and selective media. The goal is to estimate microbes present and determine if products meet BP, USP, or IP requirements.
This document describes bacterial staining techniques including Gram staining and acid-fast staining. Gram staining differentiates bacteria based on cell wall structure and stains Gram-positive bacteria purple and Gram-negative bacteria pink or red. Acid-fast staining identifies acid-fast bacteria that retain the primary stain carbolfuchsin despite decolorization. Procedures for both Gram staining and acid-fast staining of mixed bacterial samples are provided along with questions about the techniques.
The document discusses various evaluation tests performed on parenteral products, including sterility testing, clarity testing, leakage testing, pyrogen testing, and assay. Sterility testing involves incubating samples in culture media to check for microbial growth. Clarity testing examines products for visible particles. Leakage testing checks for cracks in ampoules. Pyrogen testing involves injecting products into rabbits to monitor for fever responses. Assay is performed to quantify the active ingredient in the parenteral preparation according to pharmacopeia methods. Proper testing helps ensure parenteral products are free of contaminants and contain the correct amount of active pharmaceutical ingredient.
This document provides instructions for using an ELISA kit to detect the mycotoxin zearalenone in cereal crops and animal feeds. It begins with an introduction to zearalenone and its health effects. It then describes the intended use, principle, reagents, materials, precautions, extraction procedure, and assay procedure for the zearalenone ELISA kit. The kit is designed to quantitatively detect zearalenone in samples through a competitive enzyme immunoassay.
The western blot is a technique used to detect specific proteins in a sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are sample preparation, gel electrophoresis, blotting, blocking, antibody probing, and detection. Western blotting allows researchers to identify proteins from complex mixtures and is widely used in molecular biology and medical diagnosis, such as detecting HIV, HBV, and HSV infections.
The document summarizes various physical and microbiological methods for testing semisolid dosage forms like ointments. It describes tests to evaluate rate of absorption, non-irritancy, rate of penetration, rate of drug release, viscosity, content uniformity, microbial content, and preservative efficacy. It also provides details on procedures for sterility testing using membrane filtration or direct inoculation methods.
This document provides instructions for performing an enzyme-linked immunosorbent assay (ELISA) to quantitatively detect total aflatoxins (B1, B2, G1, and G2) in grains, nuts, and other commodities. Aflatoxins are toxic metabolites produced by certain molds that can contaminate foods and animal feeds. The ELISA uses an aflatoxin-specific antibody to competitively bind sample aflatoxins or HRP-conjugated aflatoxin. The intensity of the color reaction indicates the aflatoxin concentration in samples, which can be quantified by comparison to standard curves. Validation studies showed the assay can reliably detect aflatoxins from 1-20 p
Detection techniques for microorganisms in food of animalMANJEET RATHOUR
The detection and enumeration of microorganisms in food are an essential
part of any quality control or food safety plan. Traditional methods of detecting foodborne pathogenic bacteria are often time-consuming because of the need for growth
in culture media, followed by isolation, biochemical and/or serological identifi cation,
and in some cases, subspecifi c characterization. Advances in technology have made
detection and identifi cation faster, more sensitive, more specifi c, and more convenient than traditional assays. These new methods include for the most part antibodyand DNA-based tests, and modifi cations of conventional tests made to speed up
analysis and reduce handling.
This document provides information on staining techniques used in microbiology, including the purpose, procedures, and interpretations of common staining methods. It discusses:
- The purpose of staining is to increase contrast between microorganisms and the background under the microscope.
- Procedures for simple, differential, Gram, and acid-fast (Ziehl-Neelsen) staining are described in detail, including required materials and steps.
- Interpretation of results for each stain is also explained, such as Gram-positive and Gram-negative bacteria appearing different colors.
- Preparation of smears from different specimen types and fixing methods prior to staining is covered.
This document provides an overview of immunofluorescence (IF) techniques used in dermatology. IF can be used to directly detect antigens in tissue or indirectly detect circulating antibodies in serum. It involves using fluorescently-labeled antibodies that bind to target antigens, which are then viewed under a fluorescence microscope. Direct IF is used to detect in vivo antigen deposition in skin biopsies, while indirect IF detects circulating antibodies in serum. Modifications include antigen mapping to determine structural protein localization in epidermolysis bullosa, and salt split skin techniques to differentiate subepidermal bullous disorders. IF plays a key role in diagnosing immunobullous diseases and providing insight into the pathogenic mechanisms of various skin conditions.
In situ hybridization (ISH) is a technique that uses labeled probes to localize specific DNA or RNA sequences within cells in a tissue sample. It allows researchers to obtain information about gene expression and genetic loci in their cellular context. There are two main types of ISH - fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH). ISH requires many optimization steps for each tissue and probe used but can provide insights into physiological processes and disease pathogenesis by identifying specific mRNA sequences within individual cells.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
Immunohistochemistry description of the fluorescence mehodes and enzymetic m...HadeelAlboaklah
This document defines immunohistochemistry and describes techniques for identifying cellular or tissue constituents using antigen-antibody interactions. It discusses antigens, antibodies, antibody-antigen binding, and two main methods - immunofluorescence and enzymatic. The immunofluorescence method uses fluorescent dyes to label antibodies and allow detection under a fluorescence microscope. The enzymatic method uses enzyme-labeled antibodies and reaction with a substrate to yield a colored product detectable by light microscope.
polymerase chain reaction work-flow by Dr kelvin Agimogim.pptxkelvinagimogim1
Embark on a PCR expedition! Explore the fascinating journey through the Polymerase Chain Reaction (PCR) workflow, where science seamlessly blends with precision. Unveil the mysteries of PCR, from DNA discovery to mastering amplification, presented with clarity and a touch of flair. Prepare to immerse yourself in the realm of genetic innovation, elevating your understanding of this revolutionary technique. Join us as we amplify the excitement together!
This document provides an overview of diagnostic microbiology. It discusses the goals of clinical microbiology laboratories in testing specimens to identify microorganisms causing illness and providing antimicrobial susceptibility results. It also describes various laboratory procedures used, including microscopy, culture-based techniques, immunological and molecular assays. Specimen collection, processing, staining methods, and interpretation of culture results are discussed in detail.
The document discusses diagnostic microbiology and the role of the clinical microbiology laboratory. The key responsibilities of the laboratory include testing specimens to identify microorganisms causing illness, providing antimicrobial susceptibility results, and advising physicians. Important techniques used in diagnosis include microscopy, culture, antigen detection methods like ELISA, and molecular methods like PCR. Proper specimen collection, transport, and processing are essential for accurate diagnostic testing.
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 describes techniques for isolating pure cultures of microorganisms, including serial dilution, spread plating, streak plating, and pour plating. Serial dilution involves sequentially diluting a sample to reduce the concentration of microbes and allow discrete colonies to form. Spread plating involves spreading diluted samples evenly across agar plates, streak plating uses inoculation loops to streak samples in patterns to further dilute and separate microbes, and pour plating involves mixing diluted samples into molten agar before pouring into plates. These techniques are important for isolating pure cultures needed to accurately identify and study microbes.
University Institute of Pharmaceutical Sciences is a flag bearer of excellence in Pharmaceutical education and research in the country. Here is another initiative to make study material available to everyone worldwide. Based on the new PCI guidelines and syllabus here we have a presentation dealing with the quality control tests of parenteral as referred in the pharmacopoeia.
Thank you for reading. Hope it was of help to you.
UIPS,PU team
ANTIBIOTIC SENSITIVITY TEST
Tube dilution and agar plate method.
Filter paper and cup plate method.
Ditch-plate method.
Phenol coefficient method.
Kelsey Sykes method.
This document provides instructions for using an ochratoxin A assay kit. It describes ochratoxin A as a mycotoxin produced by molds that is toxic and carcinogenic in humans and animals. The kit is intended to quantitatively detect ochratoxin A in grains, coffee, cocoa, spices and other foods. It works by competitively binding ochratoxin A from standards and samples to an antibody, and then detecting the amount of bound conjugate using a colorimetric reaction. Detailed procedures are provided for extracting ochratoxin A from different sample types and running the assay.
OBJECTIVES
Up on completion to this presentation you will be able to:
know about H pylori definition and background
Know diagnostic method of H pylori
List available serological test for H pylori with their:-
Intended use
Principle
Precaution
Limitation
Interpretation and
Quality Control
Introduction
Most common chronic bacterial infection in world. Key constituent of human micro biome.
Gram negative Spiral shape
Multiple unipolar flagella – moves freely
Produces urease
Micro-aerophillic
3 μm long with a diameter of about 0.5 μm
More common in low socioeconomic status
Humans are major reservoir Housing density, crowded conditions in the home, number of siblings, sharing a bed, and lack of hot running water
The document provides an overview of intensive care unit (ICU) training. It defines the ICU and its purpose of providing life support and monitoring for critically ill patients. It discusses types of ICUs including postoperative, pediatric, and neonatal units. Key aspects of ICU care covered include vital sign management, oxygen therapy, common procedures, scales used to assess patients, and equipment. Invasive and noninvasive monitoring techniques are also outlined.
This document defines vitamins and minerals, classifies different vitamins, discusses their sources and functions. It describes vitamin and mineral deficiencies and their symptoms. Key points include: vitamins are organic compounds necessary for health; there are two groups of vitamins - water and fat soluble; common vitamins include A, B, C, D, E and K; deficiencies can cause diseases like scurvy and rickets; minerals are inorganic elements from the earth's crust essential for body structure and processes.
This document defines vitamins and minerals, classifies different vitamins, discusses their sources and functions. It describes vitamin and mineral deficiencies and their symptoms. Key points include: vitamins are organic compounds necessary for health; there are two groups of vitamins - water and fat soluble; common vitamins include A, B complex, C, D, E and K. Minerals are inorganic elements needed for growth; important minerals are calcium, iron, iodine and phosphorus. The document provides details on specific vitamins and minerals.
Proteins are essential components of every cell and are needed for the body's structures, functions, and regulation. They are made up of amino acids and provide many critical functions. Important protein sources include meat, eggs, dairy, and beans. Digestion of proteins begins in the stomach and is completed in the small intestine. Lipids are fats and oils that provide stored energy and serve other crucial roles like insulation. Essential fatty acids must be obtained through diet as the body cannot produce them. Lipid digestion begins in the stomach and small intestine with the help of enzymes.
The document outlines a nutrition course covering topics such as carbohydrates, proteins, vitamins, minerals and water. It also discusses why health workers study nutrition and some common nutrition-related health problems. Key points include definitions of food, nutrition, diet and malnutrition. Carbohydrate digestion and metabolism are explained in detail. Causes and signs of malnutrition and malabsorption syndrome are also provided.
This document discusses different patterns of inheritance including recessive, dominant, autosomal, and sex-linked traits. It provides examples of several genetic diseases that are inherited through autosomal recessive or dominant patterns including cystic fibrosis, Gaucher disease, osteogenesis imperfecta type 1, and Huntington's disease. It describes the genes involved, typical symptoms, and inheritance patterns for each of these conditions.
This document provides an overview of hormones, including:
1. Hormones are chemicals secreted by endocrine glands that travel through the blood and influence other glands and organs. There are two major classes: protein hormones and steroid hormones.
2. The hypothalamus and pituitary gland control hormone release through negative feedback loops. The hypothalamus secretes hormones that stimulate or inhibit the pituitary gland.
3. Hormone disorders can occur if glands do not produce the proper hormones. Examples include congenital adrenal hyperplasia and androgen insensitivity syndrome.
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2. • Immunofluorescence is a serological test where the labeling of antibodies
or antigens is done with fluorescent dyes ( fluorochromes).
• Fluorochromes are dyes which have the ability to absorb the short
wavelength UV radiation and emit light of longer wavelength fluorescence (
visible green light).
• Examples : FITC, Rhodamine , Acridine orange
• First this technique was discovered by albert coons in 1942
3. • There are two ways of doing IF staining
• Direct immunofluorescence
• Indirect immunofluorescence
• Direct immunofluorescence
Use: Direct detection of Pathogens or their Ag’s in tissues or in
pathological samples
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over it
• Slide is washed to remove unattached Ab’s
• Examined under UV light in an fluorescent microscope
• The site where the Ab attaches to its specific Ag will show apple green
fluorescence
4. • DIRECT IMMUNOFLUORESCENE.
• Direct immunofluorescence (DIF) is a technique used in the laboratory to diagnose diseases of
the skin, kidney, and other organ systems. It is also called the direct immune fluorescent test
or primary immunofluorescence.
• Principle of the test
• DIF involves the application of antibody–fluorophore conjugate molecules to samples of patient
tissue obtained from biopsies.These antibody–fluorophore conjugates target
abnormal depositions of proteins in the patient’s tissue.When exposed to light,
the fluorophore emits its own frequency of light, seen with a microscope.The particular staining
pattern and type of abnormal protein deposition seen in the tissue sample help diagnose the
disease.
5.
6.
7. • What happens to the specimen in the laboratory?
1. DIF may be carried out in an automated machine or manually.The process involves first making frozen sections then
carrying out immunofluorescence.
2. Preparing frozen sections
3. A punch biopsy is transported to the lab on saline-soaked gauze.
4. The specimen is placed in a gel.
5. Liquid nitrogen is used to freeze the specimen and gel.
6. The frozen specimen is contained within the frozen gel.
7. The specimen can be stored in liquid nitrogen for about one week.
8. 4–6 micron thick slices are cut
8. • Carrying out direct immunofluorescence
1. Five or six slides are made; each for a different reagent.One slide will be used for a normal H&E stain.
2. A special pen is used to draw a perimeter to keep reagents within the slides.
3. The slides are washed.
4. The reagents are made up.
5. The reagents (antibody–fluorophore conjugates to IgG, IgM, IgA, complement protein C3, and when
required fibrinogen) are dropped onto the slides and the slides are left for some time in the dark.
6. The slides are washed again in solution.
7. Glass covers are placed over the slides.
9. • Interpretation of direct immunofluorescence.
• The prepared immunofluorescence slides are examined by a pathologist to determine
the primary sites of immune deposition (if any), the classes of immunoglobulin or other immune
deposits, and the patterns of deposition. Staining patterns can be classed into five groups:
1. Intercellular surface staining (ICS) pattern
2. Linear basement membrane zone (BMZ) pattern
3. Granular BMZ pattern
4. Shaggy BMZ pattern
5. Vascular and other patterns.
10. INDIRECT IMMUNOFLUORENCETECHNIQUES.
• A laboratory test used to detect antibodies in serum or other body fluid.The
specific antibodies are labeled with a compound that makes them glow an
apple-green color when observed microscopically under ultraviolet light.
11.
12. • Principle of the test
• For the determination of autoantibodies or antibodies against infectious agents,
cells, tissue sections or purified, biochemically characterized substances are
used as antigen substrates.
• If the sample is positive, specific antibodies in the diluted serum sample attach
to the antigens coupled to a solid phase.
• In a second step, the attached antibodies are stained with fluorescein-labelled
anti-human antibodies and visualized with the fluorescence microscope.
• Positive samples can be titrated in steps.The most suitable titration interval is
provided by the dilution factor 3.162 (square root of 10). In this way, every
second step represents in its denominator an integral power of 10 (1:10, 1:32,
1:100, 1 : 320, 1 : 1000, 1 : 3200, 1 : 10000 etc.).
13. • INDIRECT IMMUNOFLUORESCENCETEST (IIFT)
• INTRODUCTION
• Immunofluorescence is a method of using the specific reactivity of antibodies
with antigen to reveal the presence of these antibodies in sera and other body
fluids or to identify antigens in tissues in the presence of fluorescent dyes
(=fluorochromes). Immunofluorescence technique combines the sensitivity and
specificity of immunology with the precision of microscopy.
• Direct immunofluoresecence: Specific antibodies are conjugated with
fluorescent compounds.The conjugated antiserum is added to tissues and thus
fixed to the antigens. Unbound antibodies and non-antibody proteins are
removed by washing and the preparation is observed in a fluorescence
microscope.
14. • Indirect immunofluorescence: Indirect fluorescence is a double antibody
technique. the unlabelled antibodies which have bound to the antigens are
visualized by a fluorescent antiglobulin reagent directed at the unlabelled
antibodies.
1. Criteria for judging a fluorochrome as a suitable dye are:
2. The fluorochrome should posses chemical groups which will form covalent
bonds with protein molecules.
3. Easy removal of unreacted fluorescent material is also important
4. Fluorescent color of the conjugate should be different from that of the
background.
5. Conjugate should be stable under storage conditions.
15. • The fluorescence emission of FITC (Fluorescein isothiocyanate) conjugates
is green with a maximum wavelength at 529 nm.
• Fading of fluorescence:The fluorescence of microscopical preparations is
subject to fading during illumination and there may be a color change.There
should be minimum exposure to illumination during the microscopic
examination.
16. • MATERIALS
• Slides with antigen
• Positive control serum
• A positive patient's serum with a high IFT titer (at least 1:1,280) is preserved with me
thiolate (final concentration 1:10,000), divided into aliquots of 0.2 ml and kept frozen at
-20 C.
• Negative control serum
• The negative control serum should not give any fluorescence. Handle and store in the
same way as the positive control.
• PBS pH 7.4 (Check pH before use and adjust if necessary with 1N NaOH or 1N HCl)
• Conjugated anti-human globulin
17. • Use polyvalent anti-human conjugate e.g. anti-human immuno globulin (sheep) fluorescein-labeled. The working dilution of every batch
should be determined by making a series of dilutions of the dissolved conjugate.The optimal working dilution is considered to be that
dilution which is one double dilution lower than the highest dilution giving maximal fluorescence. A dilution of 1:100 is usually suitable.
1. Incubation chamber
2. A large Petri dish can be used as incubation chamber. The filter paper is placed on the bottom to keep the atmosphere humid. The slides
can be placed on glass rods.
3. Staining jar
4. Glycerin-PBS
5. Mix 9 parts glycerin (water-free, distilled) and 1 part PBS pH 7.4
6. Coverslips
7. Fluorescence microscope
18. • PREPARATION OF ANTIGEN SLIDES
• Clean the microscope slides thoroughly in hot water with a detergent or leave overnight in a
dichromate-sulfuric acid mixture.
• Rinse the slides 4 to 5 times with hot tapwater and twice with distilled water, then rub dry with a
towel.
• Put 10 drops of glycerol on the slides in two rows of 5 drops each.
• Spray the slides withTeflon in a fume cupboard to avoid inhalation.
• After drying at room temperature, rinse the slides thoroughly with lukewarm tap water and
distilled water, dry on filter paper.The slides have now been coated withTeflon, apart from the
spots where the glycerin drops were put
19. • Drop about 10 ul of a leptospira culture on theTeflon free spots. Leptospira copenhageni strain Wijnberg is
used, grown 2-3 days in EMJH medium.
• The density of the culture is considered optimal when the leptospira are lying separately in the dried drops.
If the culture is too dense, dilute the antigen with culture medium.
• Let the slides with antigen dry at room temperature on the bench. Fix the antigen on the slides by plunging
them for 10 minutes in cold acetone and dry again at room temperature.
• Now the slides are ready for use.The antigen is stable for at least 1 year at 4 C.To store the antigen, wrap
the slides in soft tissue (or soft toilet paper) and seal air tight in plastic bags.
• Before use allow the slides to come to room temperature before opening the plastic bag, to avoid
condensation of water vapor from the air on the antigen spots, which deteriorates the antigen.
20. • PERFORMANCE OFTHETEST
• Make doubling dilutions of the patients' serum in PBS, pH 7.4, starting with 1:10
up to 1:5,120.The positive serum is also diluted from 1:10 to 1:5,120.The
negative control serum is diluted from 1:10 to 1:160.
• Put 10 ul of the dilutions of patient's and control sera on the antigen spots,
starting with the highest dilution. As the negative control serum uses only half a
slide (5 antigen spots), use the other half of the slide for antigen control by
dropping 10 ul of PBS on the antigen spots.
• Incubate the slides for 30 minutes at room temperature in a humid closed Petri
dish.
• Put the slides in a staining jar with PBS and discard the PBS carefully. Add fresh
PBS. Keep for 10 minutes in PBS.
• Discard the PBS. Dry the slides around the antigen spots with soft tissue paper
and place the slides in the humid Petri dish.
• Drop 10 ul of the diluted conjugate on every antigen spot.
21. • Incubate in the dark for 30 minutes at room temperature.
• Repeat step 4 but put the staining jar in the dark, during the washing procedure.
• Repeat step 5 but place the slides in a slide-holder.
• Put a few drops of glycerin-PBS on the slide.
• Cover the slide with a coverslip, remove air bubbles by pressing softly. Wipe off
the superfluous glycerin-PBS.
• When washing the slides do not forget first to fill the jar with PBS. Avoid the
lower serum dilutions flowing over the stops with the higher dilutions.This is to
prevent false positive reactions in the higher dilutions.
• It is important to squeeze out the superfluous glycerin-PBS. A too thick
preparation gives difficulties in focussing.
22. • READING OFTHETEST
• Examine the slides under the fluorescence microscope. For fluorescence
microscopy filter combinations are dependent on the light source and the
fluorescent labels.
• The intensity of the fluorescence is read as follows:
• ++++ brilliant fluorescence
• +++ bright fluorescence
• ++ clearly visible fluorescence
• +/- weak fluorescence
• The titre of the serum is the highest dilution giving 1+ fluorescence. It is
characteristic for a positive serum that the intensity of the fluorescence
diminishes gradually in the higher dilutions