Gram-positive spherical/ovoid cocci arranged in long chains; commonly in pairs
•Non-spore-forming, nonmotile
•Can form capsules and slime layers
•Facultative anaerobes,Capnophilic
•Catalase Negative
•Most parasitic forms are fastidious and require enriched media
•Small, non pigmented colonies
•Sensitive to drying, heat, and disinfectants
This document provides information on a human orosomucoid 2 (ORM2) ELISA kit that allows for the quantitative determination of ORM2 concentrations in biological samples like serum, plasma, tissue homogenates, and cell culture supernatants. It describes the intended use, test principle, materials included in the kit, sample collection and storage recommendations, limitations of the procedure, reagent preparation instructions, and the assay procedure.
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 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.
The document describes various microbiological methods for testing samples, including total viable aerobic count, tests for specific microorganisms like Escherichia coli and Salmonella spp., and culture medium used. Glassware and equipment are sterilized using hot air oven or autoclave. Samples A-I are tested after 0, 30, or 120 days of storage. Microbial growth is observed on agar plates after incubation and used to identify microorganisms. Counts are made to determine microbial load in the samples. Serial dilution and membrane filtration methods are used to estimate total viable aerobic count.
The researchers will conduct an experiment to evaluate the potential of Golden Apple Snail meat as a culture medium nutrient source. They will gather Golden Apple Snails from rice fields and prepare both a snail meat concentrate and dried snail meat powder. Various formulations of solid and semi-solid culture media will be prepared using the snail meat products and tested for growth of selected microorganisms. Nutrient agar and SIM media will act as positive controls. Growth results will help determine the suitability of Golden Apple Snail meat as an alternative nutrient source for culture media.
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 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 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.
This document provides information on a human orosomucoid 2 (ORM2) ELISA kit that allows for the quantitative determination of ORM2 concentrations in biological samples like serum, plasma, tissue homogenates, and cell culture supernatants. It describes the intended use, test principle, materials included in the kit, sample collection and storage recommendations, limitations of the procedure, reagent preparation instructions, and the assay procedure.
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 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.
The document describes various microbiological methods for testing samples, including total viable aerobic count, tests for specific microorganisms like Escherichia coli and Salmonella spp., and culture medium used. Glassware and equipment are sterilized using hot air oven or autoclave. Samples A-I are tested after 0, 30, or 120 days of storage. Microbial growth is observed on agar plates after incubation and used to identify microorganisms. Counts are made to determine microbial load in the samples. Serial dilution and membrane filtration methods are used to estimate total viable aerobic count.
The researchers will conduct an experiment to evaluate the potential of Golden Apple Snail meat as a culture medium nutrient source. They will gather Golden Apple Snails from rice fields and prepare both a snail meat concentrate and dried snail meat powder. Various formulations of solid and semi-solid culture media will be prepared using the snail meat products and tested for growth of selected microorganisms. Nutrient agar and SIM media will act as positive controls. Growth results will help determine the suitability of Golden Apple Snail meat as an alternative nutrient source for culture media.
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 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 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.
Gel PCR/DNA fragments extraction kit(ydf) protocol v3.0 (1)BioStar Lifetech
This document provides instructions for using the HiYield Gel/PCR DNA Fragments Extraction Kit. It begins with safety precautions for handling reagents containing hazardous compounds. It then lists the contents of the kit and provides protocols for gel extraction and PCR clean up. The gel extraction protocol involves dissolving the gel slice, binding DNA to the column, washing, and eluting purified DNA. The PCR clean up protocol prepares the sample and then follows similar binding, washing, and elution steps. Troubleshooting tips are provided to address potential issues like low recovery, poor downstream performance, and difficult gel dissolution.
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.
Sterility Testing is defined as a testing which confirms that products are free from the presence of viable microorganisms. Sterility testing is very important for medical devices, pharmaceuticals, preparations, tissue materials and other materials that claim to be sterile or free from viable microorganisms.
Microbial limit tests I.P by Dr.P.srinivas cnupogu
The document describes microbial limit testing methods for pharmaceutical and cosmetic materials. There are two main methods: testing for specific pathogenic microorganisms like E. coli, Salmonella species, S. aureus, and P. aeruginosa and determining the total aerobic microbial count. The total aerobic microbial count method involves pre-treating samples depending on solubility, filtering through a membrane, incubating, and calculating microorganisms per unit weight or volume. Confirmation tests are described to detect specific microorganisms using selective agar media, biochemical tests, and colony characteristics.
This document discusses sterility testing methodology and interpretation. It describes the principles, objectives, culture media, control tests, and methods used for sterility testing. The main methods discussed are membrane filtration (Method A) and direct inoculation (Method B). It provides details on the types of media used, how the tests are performed, and considerations for interpreting the results.
Sterility testing attempts to determine if viable microorganisms are present in pharmaceutical products by testing samples from a batch. It is done after sterilization but cannot guarantee sterility of the entire batch. Major factors that can affect the results are the testing environment, culture conditions, sampling procedure, and test method used. Common test methods include membrane filtration and direct inoculation into culture media like fluid thioglycollate and soya-bean casein digest media. Observation over the incubation period can indicate if the test and batch pass or fail sterility requirements. Guidelines stress the level of assurance depends on manufacturing conditions and batch homogeneity.
This document discusses sterility testing protocols for pharmaceutical products as per Indian Pharmacopeia guidelines. It defines sterility testing as testing to confirm absence of viable microorganisms. Sterility testing is important for medical devices and preparations like ophthalmic, injections, implants etc. The test is based on principle that microorganisms will grow in nutritive media at favorable temperature. There are two methods for sterility test - membrane filtration method suitable for liquids and direct inoculation method where samples are directly inoculated to culture media. The document discusses the different culture media and quantities of samples used based on product type.
A comparative study of In-vitro Thrombolytic activity and Anti-inflammatory a...Anindya_shuvo773
The document presents research on the thrombolytic and anti-inflammatory properties of methanolic leaf extracts of Blumea lacera and Acanthus ilicifolius. In vitro assays found that the B. lacera extract had 49.27% clot lysis compared to 38.25% for A. ilicifolius, with streptokinase as the positive control at 69.34%. Anti-inflammatory assays showed the B. lacera extract inhibited protein denaturation by 64.56% versus 45.27% for A. ilicifolius, compared to the standard acetyl salicylic acid. Overall, the results indicate the leaf extracts of both plants show significant thrombolytic and anti-
This document summarizes sterility testing procedures for pharmaceutical products. Sterility testing aims to detect any viable microorganisms that may be present. Samples are inoculated into fluid thioglycollate medium, alternative thioglycollate medium, or soybean-casein digest medium and incubated with test microbes like S. aureus, C. sporogenes, P. aeruginosa, B. subtilis, A. brasiliensis or C. albicans. Tests are done using either membrane filtration or direct inoculation methods depending on the product type and volume. After incubation, the results are observed and interpreted to determine if the product passes or fails sterility requirements.
Phytochemical and acute toxicity study of leaves of artocarpus heterophyllus lampharmaindexing
This document summarizes a study on the phytochemical screening and acute toxicity of leaves from Artocarpus heterophyllus. Methanolic and aqueous extracts of the leaves were prepared and subjected to phytochemical analysis. The analysis found flavonoids, tannins, saponins, and carbohydrates present in the extracts. An acute toxicity study in mice found both extracts to be safe at a dose of 2000 mg/kg, with no signs of toxicity after 48 hours and no deaths after 14 days.
A sterility test assesses whether a pharmaceutical product is free from microorganisms. It involves incubating samples of the product in nutrient media. There are three main methods for conducting sterility tests: direct inoculation into media, membrane filtration, and adding concentrated media to products in their original containers. Two common media used are fluid thioglycollate medium and soybean-casein digest medium. Controls and appropriate sampling methods are necessary to accurately determine if a batch meets sterility requirements.
Sterility testing is performed on pharmaceutical products to detect any viable microorganisms that could contaminate the products. There are two main methods for sterility testing - membrane filtration and direct inoculation. Membrane filtration involves filtering a sample through a membrane and incubating portions of the membrane in culture media to detect any microbes. Direct inoculation adds a sample directly to culture media and incubates it. Products like injections, implants, and dressings undergo sterility testing to assure their safety before use.
This document discusses bacterial identification using API kits. It provides an overview of API, which contains identification databases for over 967 bacterial and yeast species. It then describes various API identification systems for gram negative bacteria, gram positive bacteria, anaerobes, and yeasts. The document focuses on using the API 20E kit, outlining the materials needed, inoculation procedure, incubation, result interpretation, and limitations. It emphasizes that API kits allow for rapid identification of known bacterial species contained within their databases.
The document provides details about Narjis Fatima's internship at National Foods Limited from May to June 2016. It includes:
1) An introduction to National Foods Limited, which started as a spice company in 1970 and has grown to one of the largest food companies in Pakistan with over 110 products.
2) A description of the equipment used in the microbiology lab such as an autoclave, incubator, biosafety cabinet, and pH meter.
3) An overview of the daily activities performed in the microbiology lab including media preparation, sample testing using methods like total plate count, and sanitizing equipment.
This document describes sterility testing procedures for parenteral products. Sterility tests are performed under aseptic conditions on random samples from batches to check for any living microorganisms. Samples are incubated in culture media at specified temperatures and times to detect any microbial growth. Direct inoculation and membrane filtration methods are commonly used to transfer samples to culture media like fluid thioglycollate, soybean-casein digest, and tryptic soya broth. The sterility and growth promotion of media lots are also tested. Specific quantities of products are tested based on container size, and interpretation of results determines if a product passes or fails sterility testing.
This document summarizes a dissertation report submitted by Ashish Diwakar on microbial limit testing conducted at IPCA Laboratories Ltd. in Ratlam, Madhya Pradesh, India. It provides an introduction to IPCA and the principles and requirements of microbial limit testing. Methods for total bacterial count, total fungal count, and testing for pathogens like E. coli, Salmonella, P. aeruginosa, and S. aureus are described. Both direct inoculation and membrane filtration methods are covered. Requirements include various culture media, glassware, and equipment. Test procedures and observations are outlined.
This document provides instructions for several microbiology laboratory techniques and experiments. It includes procedures for preparing common microbiology media like nutrient broth, nutrient agar, and YPD agar. It also describes how to conduct experiments like methylene blue degradation, which is a photocatalytic reaction, and a protein cross-linking method using transglutaminase. The document provides detailed steps for culturing E. coli, using equipment like autoclaves, laminar flow hoods, and micropipettes, and following sterilization and contamination prevention protocols in the laboratory.
The document provides information about basic handling of microbiology experiments. It discusses various laboratory equipment used such as autoclave, laminar air flow, incubator, and micropipette. It also describes procedures for media preparation including nutrient broth, nutrient broth agar, yeast peptone dextrose broth and agar. Culture preparation techniques for E. coli and S. cerevisiae are outlined. Methods for methylene blue degradation and cross-linking of polymers using transglutaminase are also summarized.
The document describes procedures for testing the sterility of pharmaceutical products. It provides details on culture media, incubation temperatures, strains of test microorganisms, and the sterility test method. The key points are:
- Two common culture media are described for detecting bacteria (Fluid Thioglycollate Medium) and fungi/bacteria (Soybean-Casein Digest Medium).
- Samples are inoculated into media and incubated at specified temperatures, then examined for microbial growth which would indicate a failed sterility test.
- The sterility test method and number of samples tested depends on the type and amount of product available for testing.
This document provides information on Staphylococcus aureus, including its characteristics, habitats, infections it can cause, and virulence factors. It also describes the ISO 6888-1 standard method for enumerating coagulase-positive staphylococci (S. aureus and other species) from food and animal feed samples. The method involves inoculating Baird-Parker agar plates with sample dilutions, incubating plates at 35-37°C, selecting typical and atypical colonies, confirming colonies with a coagulase test, and calculating bacterial counts. Key components of the Baird-Parker medium and preparation steps are also outlined.
Gel PCR/DNA fragments extraction kit(ydf) protocol v3.0 (1)BioStar Lifetech
This document provides instructions for using the HiYield Gel/PCR DNA Fragments Extraction Kit. It begins with safety precautions for handling reagents containing hazardous compounds. It then lists the contents of the kit and provides protocols for gel extraction and PCR clean up. The gel extraction protocol involves dissolving the gel slice, binding DNA to the column, washing, and eluting purified DNA. The PCR clean up protocol prepares the sample and then follows similar binding, washing, and elution steps. Troubleshooting tips are provided to address potential issues like low recovery, poor downstream performance, and difficult gel dissolution.
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.
Sterility Testing is defined as a testing which confirms that products are free from the presence of viable microorganisms. Sterility testing is very important for medical devices, pharmaceuticals, preparations, tissue materials and other materials that claim to be sterile or free from viable microorganisms.
Microbial limit tests I.P by Dr.P.srinivas cnupogu
The document describes microbial limit testing methods for pharmaceutical and cosmetic materials. There are two main methods: testing for specific pathogenic microorganisms like E. coli, Salmonella species, S. aureus, and P. aeruginosa and determining the total aerobic microbial count. The total aerobic microbial count method involves pre-treating samples depending on solubility, filtering through a membrane, incubating, and calculating microorganisms per unit weight or volume. Confirmation tests are described to detect specific microorganisms using selective agar media, biochemical tests, and colony characteristics.
This document discusses sterility testing methodology and interpretation. It describes the principles, objectives, culture media, control tests, and methods used for sterility testing. The main methods discussed are membrane filtration (Method A) and direct inoculation (Method B). It provides details on the types of media used, how the tests are performed, and considerations for interpreting the results.
Sterility testing attempts to determine if viable microorganisms are present in pharmaceutical products by testing samples from a batch. It is done after sterilization but cannot guarantee sterility of the entire batch. Major factors that can affect the results are the testing environment, culture conditions, sampling procedure, and test method used. Common test methods include membrane filtration and direct inoculation into culture media like fluid thioglycollate and soya-bean casein digest media. Observation over the incubation period can indicate if the test and batch pass or fail sterility requirements. Guidelines stress the level of assurance depends on manufacturing conditions and batch homogeneity.
This document discusses sterility testing protocols for pharmaceutical products as per Indian Pharmacopeia guidelines. It defines sterility testing as testing to confirm absence of viable microorganisms. Sterility testing is important for medical devices and preparations like ophthalmic, injections, implants etc. The test is based on principle that microorganisms will grow in nutritive media at favorable temperature. There are two methods for sterility test - membrane filtration method suitable for liquids and direct inoculation method where samples are directly inoculated to culture media. The document discusses the different culture media and quantities of samples used based on product type.
A comparative study of In-vitro Thrombolytic activity and Anti-inflammatory a...Anindya_shuvo773
The document presents research on the thrombolytic and anti-inflammatory properties of methanolic leaf extracts of Blumea lacera and Acanthus ilicifolius. In vitro assays found that the B. lacera extract had 49.27% clot lysis compared to 38.25% for A. ilicifolius, with streptokinase as the positive control at 69.34%. Anti-inflammatory assays showed the B. lacera extract inhibited protein denaturation by 64.56% versus 45.27% for A. ilicifolius, compared to the standard acetyl salicylic acid. Overall, the results indicate the leaf extracts of both plants show significant thrombolytic and anti-
This document summarizes sterility testing procedures for pharmaceutical products. Sterility testing aims to detect any viable microorganisms that may be present. Samples are inoculated into fluid thioglycollate medium, alternative thioglycollate medium, or soybean-casein digest medium and incubated with test microbes like S. aureus, C. sporogenes, P. aeruginosa, B. subtilis, A. brasiliensis or C. albicans. Tests are done using either membrane filtration or direct inoculation methods depending on the product type and volume. After incubation, the results are observed and interpreted to determine if the product passes or fails sterility requirements.
Phytochemical and acute toxicity study of leaves of artocarpus heterophyllus lampharmaindexing
This document summarizes a study on the phytochemical screening and acute toxicity of leaves from Artocarpus heterophyllus. Methanolic and aqueous extracts of the leaves were prepared and subjected to phytochemical analysis. The analysis found flavonoids, tannins, saponins, and carbohydrates present in the extracts. An acute toxicity study in mice found both extracts to be safe at a dose of 2000 mg/kg, with no signs of toxicity after 48 hours and no deaths after 14 days.
A sterility test assesses whether a pharmaceutical product is free from microorganisms. It involves incubating samples of the product in nutrient media. There are three main methods for conducting sterility tests: direct inoculation into media, membrane filtration, and adding concentrated media to products in their original containers. Two common media used are fluid thioglycollate medium and soybean-casein digest medium. Controls and appropriate sampling methods are necessary to accurately determine if a batch meets sterility requirements.
Sterility testing is performed on pharmaceutical products to detect any viable microorganisms that could contaminate the products. There are two main methods for sterility testing - membrane filtration and direct inoculation. Membrane filtration involves filtering a sample through a membrane and incubating portions of the membrane in culture media to detect any microbes. Direct inoculation adds a sample directly to culture media and incubates it. Products like injections, implants, and dressings undergo sterility testing to assure their safety before use.
This document discusses bacterial identification using API kits. It provides an overview of API, which contains identification databases for over 967 bacterial and yeast species. It then describes various API identification systems for gram negative bacteria, gram positive bacteria, anaerobes, and yeasts. The document focuses on using the API 20E kit, outlining the materials needed, inoculation procedure, incubation, result interpretation, and limitations. It emphasizes that API kits allow for rapid identification of known bacterial species contained within their databases.
The document provides details about Narjis Fatima's internship at National Foods Limited from May to June 2016. It includes:
1) An introduction to National Foods Limited, which started as a spice company in 1970 and has grown to one of the largest food companies in Pakistan with over 110 products.
2) A description of the equipment used in the microbiology lab such as an autoclave, incubator, biosafety cabinet, and pH meter.
3) An overview of the daily activities performed in the microbiology lab including media preparation, sample testing using methods like total plate count, and sanitizing equipment.
This document describes sterility testing procedures for parenteral products. Sterility tests are performed under aseptic conditions on random samples from batches to check for any living microorganisms. Samples are incubated in culture media at specified temperatures and times to detect any microbial growth. Direct inoculation and membrane filtration methods are commonly used to transfer samples to culture media like fluid thioglycollate, soybean-casein digest, and tryptic soya broth. The sterility and growth promotion of media lots are also tested. Specific quantities of products are tested based on container size, and interpretation of results determines if a product passes or fails sterility testing.
This document summarizes a dissertation report submitted by Ashish Diwakar on microbial limit testing conducted at IPCA Laboratories Ltd. in Ratlam, Madhya Pradesh, India. It provides an introduction to IPCA and the principles and requirements of microbial limit testing. Methods for total bacterial count, total fungal count, and testing for pathogens like E. coli, Salmonella, P. aeruginosa, and S. aureus are described. Both direct inoculation and membrane filtration methods are covered. Requirements include various culture media, glassware, and equipment. Test procedures and observations are outlined.
This document provides instructions for several microbiology laboratory techniques and experiments. It includes procedures for preparing common microbiology media like nutrient broth, nutrient agar, and YPD agar. It also describes how to conduct experiments like methylene blue degradation, which is a photocatalytic reaction, and a protein cross-linking method using transglutaminase. The document provides detailed steps for culturing E. coli, using equipment like autoclaves, laminar flow hoods, and micropipettes, and following sterilization and contamination prevention protocols in the laboratory.
The document provides information about basic handling of microbiology experiments. It discusses various laboratory equipment used such as autoclave, laminar air flow, incubator, and micropipette. It also describes procedures for media preparation including nutrient broth, nutrient broth agar, yeast peptone dextrose broth and agar. Culture preparation techniques for E. coli and S. cerevisiae are outlined. Methods for methylene blue degradation and cross-linking of polymers using transglutaminase are also summarized.
The document describes procedures for testing the sterility of pharmaceutical products. It provides details on culture media, incubation temperatures, strains of test microorganisms, and the sterility test method. The key points are:
- Two common culture media are described for detecting bacteria (Fluid Thioglycollate Medium) and fungi/bacteria (Soybean-Casein Digest Medium).
- Samples are inoculated into media and incubated at specified temperatures, then examined for microbial growth which would indicate a failed sterility test.
- The sterility test method and number of samples tested depends on the type and amount of product available for testing.
This document provides information on Staphylococcus aureus, including its characteristics, habitats, infections it can cause, and virulence factors. It also describes the ISO 6888-1 standard method for enumerating coagulase-positive staphylococci (S. aureus and other species) from food and animal feed samples. The method involves inoculating Baird-Parker agar plates with sample dilutions, incubating plates at 35-37°C, selecting typical and atypical colonies, confirming colonies with a coagulase test, and calculating bacterial counts. Key components of the Baird-Parker medium and preparation steps are also outlined.
Sterilisation-
Is the process of making something free from bacteria or other living microorganisms.
Sterility Testing-
Are done to detect if viable forms of micro-organisms are present or not on or in the pharmaceutical preparations.
B12 CULTURE AGAR (L-LEICHMANNII MAINTANCE MEDIUM)- Dehydrated Culture Mediacdhfinechemical
B12 Culture Agar is recommended for the propagation, cultivation and maintenance of Lactobacillus leichmannii in the Vitamin B12 Assay. Us 7830 used as the test organism.
This document is a lab report from a student at the University of Zambia describing an experiment to prepare routine diagnostic media and isolate bacteria. The student prepared two types of media - blood agar and MacConkey agar - by diluting commercial powder in distilled water and autoclaving the mixtures. Samples of milk and intestinal washing were then streaked onto the prepared plates and incubated at 37 degrees Celsius overnight. The following day, bacterial growth was observed on the plates, indicating the prepared media supported bacterial culture successfully.
Production of secondary metabolites : enzymes which involves the upstream technological process
Introduction
History
Process involved
Contribution of different micro-organisms
Flowchart
Example: Methods Production of Amyalse in industrial view
IntroductionIn terms of total production tonnages used f.docxvrickens
Introduction
In terms of total production tonnages used for food, wheat is currently second to rice as the main human food crop and is the leading source of vegetable protein in human nutrition (Nutrient Data Laboratory). Aphids (Order Hemiptera) are major insect pests of world agriculture, damaging crops by removing photoassimilates and vectoring numerous plant viruses (Smith and Boyko, 2007). The grain aphid (Sitobion avenae) is considered a serious pest of commercial wheat in the UK. Many aphid species can develop resistance to insecticides (Devonshire and Field, 1991), and restrictions on the availability of active ingredients for insecticide production in Europe (European Directives 91/414/EEC) has prioritized research on crop varieties with resistance to aphid pests in UK agriculture (Painter, 1951; Panda and Kush, 1995; Smith, 2005). Most commercial wheat varieties have very little resistance to aphid pests (Lee, 1984; Dedryver and Di Pietro, 1984; Di Pietro and Dedryver, 1986; Migui, 2002; Migui and Lamb, 2003), with at best partial antibiosis, antixenosis and tolerancein some winter varieties (Lowe, 1984a; Lowe, 1984b; Havlícková, 1993). Wheat genetics are more complicated than that of most other crop species. Some wheat species are diploid, with two sets of chromosomes, but many are stable polyploids, with four sets of chromosomes (tetraploid) or six (hexaploid). Modern wheat varieties grown in the U.K. are hexaploid and have low genetic diversity for insect resistance traits (Ferry et al, 2011; Ogbonnaya et al, 2013; niab.com).
Diploid wheat (T. monococcum) lines have been observed to exhibit high levels of resistance against S. avenae (Migui and Lamb, 2003; 2004; Ferry et al, 2011). However, no previous studies have been carried out in these lines to investigate differentially expressed genes in response to aphid infestation. Therefore, differential proteomic analysis is being employed to identify putative defence responses in diploid wheat lines (Triticum monococcum L.) when subjected to grain aphid (S. avenae) feeding in order to better understand the basis of this observed resistance/tolerance.
In this lab you will conduct a 2D-electrophoresis separation of the wheat leaf proteome.
Methods
It is essential that you retrieve the full lab manual produced by GE healthcare for 2D electrophoresis:
Step 1 - Sample Solubilization
This has been done for you.
In this experiment 200mg of wheat leaf was ground in liquid nitrogen to a fine powder. The protein pellets available to you have been incubated in 10% (w/v) trichloroacetic acid/ acetone with 0.07% v/v 2-mercaptoethanol at -20 °C for 16 hours and then centrifuged at 35 000 x g for 20 mins.
> The pellets were washed with ice-cold acetone (0.07% 2-mercaptoethanol) 4-6 times and finally incubated at -20 °C for 1 h, and centrifuged at 12 000 x g for 15 min.
>>An acetone wash using 1 part sample: 3 parts acetone (w/v) was performed, the sample was vortexed to disperse pelle ...
Media preparation involves defining media as the nutrients used to culture microorganisms. Media can be solid or liquid, and synthetic or non-synthetic depending on ingredients. Solid media like agar is used to isolate bacteria and observe colony characteristics. Liquid media like broth is used to propagate large numbers of organisms and conduct fermentation studies. Quality control of media includes checking for proper growth, physical characteristics, gel strength and contamination. Media must be stored under controlled conditions and used before the expiry date to ensure quality.
1. PEA agar is a selective culture medium that allows growth of gram-positive bacteria like Staphylococcus while inhibiting most gram-negative bacteria.
2. PEA contains phenyl ethyl alcohol which disrupts the membranes of gram-negative bacteria.
3. PEA agar is used to isolate gram-positive bacteria from mixed samples and to inhibit common contaminants like E. coli and Proteus species.
Biol 390-Lab 3 Introduction to the Bioreactor
Maryville University
Objective Become familiar with a lab scale bioreactor
Background Bioreactors are vessels that support the growth of single-celled prokaryotic or eukaryotic
cells. They can be used to increase the biomass of an organism, such as bakers yeast. In
other cases the biomass of an organism is increased, then used to convert a pre-cursor to
a product, such as in penicillin production. Fermentations can be either aerobic or
anaerobic depending on the organism.
Vessels can be made of glass in small-scale bioreactor. In large scale reactors are
typically made of stainless steel
Overview a. BioFlo/CelliGen 115 is a versatile fermentor/bioreactor that provides a fully
equipped system in a compact package. It can be employed for batch, fed batch,
or continuous culture with process control for pH, dissolved oxygen (DO),
agitation, temperature, pump feed, antifoam and foam/level.
_______________________________________________
2
Functions • Microbial metabolism and growth can be controlled though
o Agitation speed and type of agitator
o Temperature 20oC over coolant temperature and up to 70oC
o Aeration - Four gases can be introduced and their flow controlled:
§ Air
§ Nitrogen
§ Carbon dioxide
§ Oxygen
o pH can be controlled between 2 and 14 using a pH sensor. Control is maintained by
a P&I (proportional and integral) controller, which operates the acid and base
pumps.
o Dissolved oxygen can be controlled in the range of 0 to 200%. An electrode senses
D.O and levels can be controlled by a P&I controller.
o Foam control – A foam/level sensor is present in the head-plate. The controller
operates an antifoam pump. Pumps turn on and off.
o Exhaust system – gases and moisture are removed.
o Sampler – samples can be taken into a sample tube by closing the exhaust.
Headplate
3
Touch Screen This has a summary screen for control of
o Agitation
o Temperature
o pH
o DO
o Air
o Summary screen
o Gauge screen
4
Setpoint Screen
Before you start 1. Connect water to the system and turn it on.
2. Make sure the drain line is properly connected to the system.
1. Note we currently not using a water cooling system
3. Connect the quick-connect plastic water lines to the exhaust condenser.
4. Add glycerin to the thermowell and insert the temperature probe.
5. Make sure the motor is not connected. Turn the mains/power ON.
6. Set the TEMP setpoint to the desired working temperature.
7. Check that agitation (Agit) is in OFF mode. Connect the motor, then set
5
agitation to the desired speed, and select Auto as its control mode.
8. Remove the shorting cap from the pH probe. Connect the pH cable to the pH
probe.
9. Remove the protective cap from the DO probe and connect the DO cable to the
DO probe.
10. If you have a water-jacket.
This document provides guidance on performing a western blot experiment, including sample preparation, gel electrophoresis, protein transfer, antibody incubation, and detection. The key steps are:
1. Extracting and quantifying proteins from tissue or cell samples using homogenization and centrifugation with RIPA lysis buffer.
2. Separating proteins by size using SDS-PAGE gel electrophoresis, then transferring them to a membrane for antibody detection.
3. Incubating the membrane with primary and secondary antibodies, then using chemiluminescence or DAB to detect bound antibodies and visualize protein bands.
4. Analyzing protein bands using imaging software to identify target proteins.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
mô tả các thí nghiệm về đánh giá tác động dòng khí hóa sau đốt
B3 07 1
1. 3.7 Reagents, Media, and Media Preparation - 1
2004
3.7 Reagents, Media, and Media Preparation
A. Growth Media
Most bacteriological culture medias are commercially available as pre-made or as bases, which can be
easily prepared in the laboratory. These commercial products are entirely acceptable and should be
made and stored according to the manufacturer’s recommendations. Recipes for those media which
are not available in a commercial preparation are included below.
1. Brain Heart Infusion Agar (BHIA) (Difco 1998)
A basic agar for most bacterial cultures.
2. Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB) (Difco 1998)
A basic media for most bacterial cultures.
3. Selective Kidney Disease Medium-2 (SKDM-2) (Austin et al. 1983)
A media used for selective isolation of Renibacterium salmoninarum.
Peptone 10g
Yeast extract 0.5g
L-Cysteine HCL 1g
Agar 15g
Distilled water 874mL
Adjust pH to 6.5 before adding agar. Autoclave for 15 minutes at 121o
C. Cool to ~ 50o
C and add:
Fetal Bovine Serum 100.0 mL
The following antibiotics are added to reduce overgrowth from other bacterial organisms):
4.0 mL Cycloheximide (1.2 g Cyclohexamide in 96 mL dH2O)
1.0 mL D-Cycloserine (0.3 g D-Cycloserine in 24 mL of dH2O)
2.0 mL Polymyxin B-sulfate (0.3 g Polymyxin B-sulfate in 24 mL of dH2O)
1.0 mL Oxolinic acid (0.06 g Oxolinic acid in 24 mL of 5% NaOH)
B. Media to Identify Growth and Biochemical Characteristics
Most of these media are commercially available as pre-made or as bases, which can be easily
prepared in the laboratory. These commercial products are entirely acceptable and should be made
and stored according to the manufacturer’s recommendations. Unless otherwise specified, these
media can also be prepared from basic laboratory ingredients according to recipes found in the
following references: MacFaddin 1980, Difco 1998, and MacFaddin 2000.
2. 3.7 Reagents, Media, and Media Preparation - 2
2004
1. Motility Test Medium
A semi-solid media used as a tube test to detect the ability of a microorganism to exhibit motility.
Several types are commercially available, including MIO (motility, indole, ornithine) which
allows for the detection of motility, and the reaction of two biochemical tests in the same tube.
2. Trytic Soy Broth (TSB)
A nutrient broth media used to determine motility of a microorganism with the hanging drop
method.
3. Triple Sugar Iron Agar (TSI)
A commercially prepared dehydrated media used to evaluate the utilization of glucose and two
additional carbohydrates, as well as the production of hydrogen sulfide.
4. Oxidation/Fermentation (OF) Medium
A basal media for carbohydrate utilization tests, available in a commercially prepared dehydrated
powder. The OF basal is prepared according to manufacturer’s recommendations prior to the
addition of individual carbohydrates as described below:
a. To prepare final medium aseptically add 10 mL of a filter-sterilized (0.45 µm) 10%
carbohydrate solution to autoclaved and cooled (50o
C) media resulting in a 1% final
concentration, with the exception of salicin, which should be made as a 5% solution resulting
in a 0.5% final concentration (see below). Only one carbohydrate is added to the basal
medium for each test to be run.
10% Arabinose (1 g Arabinose to 10 mL in dH2O)
10% Rhamnose (1 g Rhamnose to 10 mL in dH2O)
10% Sucrose (1 g Sucrose to 10 mL in dH2O)
10% Sorbitol (1 g Sorbitol to 10 mL in dH2O)*
10% Maltose (1 g Maltose to 10 mL in dH2O)
10% Glucose (1 g Glucose to 10 mL in dH2O)
5% Salicin (0.5 g Salicin to 10 mL in dH2O)
b. Mix flask thoroughly and aseptically dispense into sterile tubes. Store at 2 to 8°C. Final pH
= 6.8 + 0.2 at 25°C.
*A sorbitol utilization slant media can also be prepared and utilized as described in Cipriano
and Pyle (1985).
5. Nutrient Gelatin
A dehydrated medium for determining the presence of Gelatinase.
6. Tryptone Broth
For use with the indole test.
Tryptone 10 g
Distilled water 1000 mL
Heat gently to dissolve. Dispense into tubes. Autoclave at 15 pounds pressure for 15 minutes and
allow tubes to cool. Store at 2 to 8°C.
7. Decarboxylase Medium Base
A basal media for use in Lysine test. The basal media, without addition of lysine, serves as the
control.
3. 3.7 Reagents, Media, and Media Preparation - 3
2004
To make L-Lysine media add 5 g L-Lysine to 1 liter of prepare basal decarboxylase media.
Dispense into tubes. Autoclave at 15 pounds pressure for 15 minutes and allow tubes to cool.
Final pH = 6.8 + 0.2 at 25°C. Store at 2 to 8°C.
8. Malonate Broth
A media used for the malonate test.
9. Bile Esculin Agar
A commercially prepared, dehydrated medium used to determine a bacterium’s ability to
hydrolyze esculin into glucose and esculetin.
C. Media Preparation
1. Plate Media
a. Prepare media in stainless steel beakers or clean glassware according to manufacturer
instructions. Check pH and adjust if necessary. Media must be boiled for one minute to
completely suspend agar. Use of a stir bar will facilitate mixing of agar.
b. Cover beaker with foil or pour into clean bottles being sure to leave lids loose. Sterilize
according to manufacturer’s instructions when given, or at 121o
C for 15 minutes at 15 pounds
pressure (consult with the manual provided by the autoclave manufacturer for adjustment of
time when large volumes of media are being sterilized).
c. Cool media to 50°C.
d. Alternatively, media can be autoclaved and cooled to room temperature and refrigerated for
later use. Store bottles labeled with media type, date, and initials. When media is needed,
boil, microwave, or use a water bath to completely melt the agar. Cool to 50°C.
e. Before pouring media, disinfect hood or counter thoroughly and place sterile petri dishes on
the disinfected surface. Mix any added ingredients into the media at this temperature.
f. Label the bottom of each plate with medium type and date prepared.
g. Remove bottle cap and pour plates or dispense with a sterile Cornwall pipette, lifting each
petri dish lid as you go. Pour approximately 15 to 20 mL per 100 X 15mm petri dish.
Replace lids as soon as the plate is poured.
h. Invert plates when the media has cooled completely (~ 30 to 60 minutes) to prevent excessive
moisture and subsequent condensation on the plate lid.
i. Allow plates to sit overnight at room temperature. Store plates upside down in the refrigerator
in a tightly sealed plastic bag or plate storage tin.
j. Follow manufacturer’s recommendation for storage period of prepared media. Each batch
should be labeled with the date of preparation.
4. 3.7 Reagents, Media, and Media Preparation - 4
2004
2. Tube Media
a. Prepare media in stainless steel beakers or clean glassware according to manufacturer’s
instructions. If the medium contains agar, boil for one minute to completely suspend the
agar. Use of a stir bar will facilitate mixing of agar.
b. Media with indicators must be pH adjusted. This can be done when medium is at room
temperature; otherwise, compensation for temperature needs to be made.
c. Arrange test tubes in racks. Disposable, autoclavable, screw cap tubes can be used for all
tube media.
d. Use an automatic pipetter or Pipette-aid™ to dispense the medium. If using the Brewer or
Cornwall pipette, prime with deionized water, then pump the water out of the syringe prior to
pipetting and discard the first few tubes of media that are dispensed. Dispense approximately
5 to 10 mL media in 16 X 125 mm or 20 X 125 mm tubes. Close caps loosely.
e. Immediately after use, rinse the automatic pipetter in hot tap water followed by distilled water
to remove all media and prevent clogging of the instrument.
f. Loosely place screw caps on tubes. Do not tighten caps. It is necessary to allow pressure to
release from tubes while heating in the autoclave.
g. Follow manufacturer’s recommendation for autoclave time and temperature.
h. If making slants, put tubes in slant racks after autoclaving. Adjust the slant angle to achieve
the desired slant angle and butt length (i.e. long butt and short slant for TSI or a standard slant
over ¾ of the tube length for TSA or BHIA). Then tighten caps.
i. Cool completely to room temperature in the slanted position.
j. Label the tubes or the tube rack with type of medium and date made.
k. Store at 2 to 8°C, following manufacturer’s recommendation for period of long-term storage.
D. Reagents
Most of these reagents are commercially available pre-made. These commercial products are entirely
acceptable and should be stored according to the manufacturer’s recommendations. The formulations
provided below were obtained from the references cited.
1. Gram Stain Reagents
These stains can be ordered as a complete kit or can be prepared as follows:
a. Crystal Violet
Crystal violet (90% dye content) 20.0 g
Ethanol (95%) 200 mL
Ammonium oxalate 8.0 g
dH20 800 mL
Mix first two ingredients and let sit overnight or until dye goes into solution. Add remaining
5. 3.7 Reagents, Media, and Media Preparation - 5
2004
ingredients and filter before use.
b. Gram's Iodine
Iodine crystals 1.0 g
Potassium iodide 2.0 g
dH20 300 mL
c. De-Colorizer
Acetone 40 mL
Ethanol (95%) 60 mL
d. Safranin
Safranin O 2.5 g
95% ethanol 100 mL
dH20 900 mL
Filter safranin solution before use.
2. FAT Mounting Fluid (pH 9.0)
Glycerol 90.0 mL
DABCO* 2.5 g
PBS 10.0 mL
Suspend the DABCO in glycerol over low heat. Then add 1X PBS (see below). Adjust pH to 8.6
to 9.0 with 1N hydrochloric acid or 0.1N sodium hydroxide. The pH of the mounting media is
important, as an acid pH will quench fluorescence. Check the pH frequently. Store at room
temperature.
* Optional ingredient - DABCO is 1,4-diazabicyclo-(2,2,2)-octane. Its addition to mounting fluid
can reduce quenching of fluorescence.
3. Phosphate-Buffered Saline for FAT (PBS), pH 7.1
a. 1x concentration (0.15 M NaCl, 0.01 M phosphate; makes 1 L)
NaCl 8.50 g
Na2HPO4 (anhydrous) 1.07 g
NaH2PO4
.
H2O (monohydrate) 0.34 g
DH2O to 1 L
Adjust pH to 7.1 with 1N hydrochloric acid or 0.1N sodium hydroxide.
b. 5x concentration (makes 10 L of 1x PBS )
NaCl 85.00 g
Na2HPO4 (anhydrous) 10.70 g
NaH2PO4
.
H2O (monohydrate) 3.45 g
DH2O to 2 L
Adjust pH to 7.1 with 1N hydrochloric acid or 0.1N sodium hydroxide.
4. Kovac’s Indole Reagent
Isoamyl alcohol 30 mL
6. 3.7 Reagents, Media, and Media Preparation - 6
2004
p-Dimethyl aminobenzaldehyde 2 g
Hydrochloric acid (HCl) 10mL
Dissolve the aldehyde in the alcohol. Slowly add the acid to the mixture. Store solution at 2 to
8°C in amber dropper bottle.
5. Counter Stains
a. Rhodamine
Rehydrate the rhodamine to 1 mg/mL in distilled water. If the rhodamine does not
completely dissolve, add a small drop of 0.1 M sodium hydroxide. Store at 2 to 8°C.
b. Evans Blue
Prepare 0.1% stock solution with distilled water and decontaminate with 0.45 µm membrane
filtration. Store at room temperature. Prepare a 0.01% working dilution with sterile PBS
(Section 2, 3.7.D.3 “Phosphate-Buffered Saline for FAT (PBS), pH 7.1”) (Cvitanich 1994).
c. Eriochrome Black T
Prepare a solution at 1:60 (w/v) in PBS (Section 2, 3.7.D.3 “Phosphate-Buffered Saline for
FAT (PBS), pH 7.1”), and filter through Whatman #1 and Whatman # 42 filter papers before
initial use (Elliott & McKibben 1997).
6. FITC Conjugated Rabbit Anti-X /Rhodamine Counter Stain
Rhodamine stock 10.0 µL
FITC conjugated antibodies 10.0 µL
Phosphate buffered saline 480.0 µL
Store at 4°C.
Note: Evans Blue or Eriochrome Black T can also be used as counterstain in FAT in a separate
step during the staining process, but neither can be added directly to the conjugate solution.
E. Cytochrome Oxidase Spot Test
Individual test strips can be purchased from several suppliers (Catalogue # 38-191, Remel, Tel. 800-
255-6730).
F. Determination of Antiserum and Conjugate Working Dilutions for FAT
Commercially prepared anti-sera and conjugates should be reconstituted according to the
manufacturer's instructions. Aliquots of 0.5 mL can be frozen for later dilution into a working
solution of the reagent. Reagents are more stable if frozen as a stock solution. The proper working
dilution is the highest dilution that still maintains maximum brightness of the fluorochrome on
positive control FAT preparations. Generally, the manufacturer will recommend between 1:20 to 1:50
using a suitable buffer (PBS) as the diluent. However, in all cases, each laboratory must establish the
proper working dilution by starting with the manufacturer’s recommendation and bracketing, or
testing dilutions on either side of the recommended concentration. The following example shows
how to determine the correct working dilution of FAT conjugate where the manufacturer recommends
a working dilution of 1:40.
7. 3.7 Reagents, Media, and Media Preparation - 7
2004
1. Using the stock solution, dilute the antiserum at 1:20, 1:30, 1:40, 1:50, 1:60 using PBS or another
buffer as recommended by antibody manufacturer.
2. Rhodamine counter stain is added directly to the optimum antiserum working dilution at a 1:50
(alternatively, Evans Blue may be used to counterstain FAT stained slides – do not add Evans
Blue to the conjugate directly).
3. The FAT is performed on replicates of a known positive control, each replicate using a different
dilution of the conjugated antiserum. In this way, the working dilution can be determined as the
endpoint of optimum fluorescence (the highest dilution that still provides a bright specific
fluorescence with little or no background staining).
4. Prepared conjugate solutions should be filtered through 0.2 to 0.4 micron filter prior to use and
storage. Store frozen in small aliquots. Do not thaw and re-freeze antibodies repeatedly.
G. PCR Reagents
The following are formulations for extraction reagents used specifically in the protocols described in
Section 2, 3.5.B.2 “Nested Polymerase Chain Reaction (PCR) for Confirmation of R. salmoninarum
DNA.”
1. Lysozyme Lysis Buffer
100 mL (this formulation is used for lysis of gram positive bacteria with Qiagen extraction kits).
Lysozyme 2 g
Tris HCl Stock 2 mL
EDTA stock 2 mL
Triton 1.2 mL
Bring these components to 100 mL with sterile distilled water (molecular grade).
2. Tris HCl Stock Solutions
100 mL at 1M pH 8.0 (for use in lysozyme buffer).
Trizma base 5.7 g
Tris HCl 8.9 g
dH2O 85.4 mL
3. EDTA Stock Solution
100 mL at 0.1 M (for use in lysozyme buffer).
EDTA 3.72 g
Bring to 100 mL with sterile distilled water (molecular grade).