DIALISIYS
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This document discusses several steps in the partial purification of proteins from cells, including: 1. Separation of proteins from cells through rupture of the cell membrane using chemicals or physical methods. 2. Precipitation of proteins from cell extracts using ammonium sulfate, which is done at 4°C overnight at a concentration based on the salt's saturation level. 3. Dialysis of the precipitated proteins against a buffer such as PBS to remove the ammonium sulfate before further purification steps.
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Biochemical tests for characterization of bacteria
The document describes various biochemical tests used to characterize bacteria, including the IMViC tests, oxidase test, gelatin hydrolysis test, and starch hydrolysis test. The IMViC tests include indole, methyl red, Voges-Proskauer, and citrate tests used to identify members of the Enterobacteriaceae family. The oxidase test detects the enzyme cytochrome c oxidase to distinguish between oxidase-positive and -negative bacteria. The gelatin hydrolysis test identifies bacteria that can produce the protease gelatinase to liquefy gelatin. The starch hydrolysis test detects bacteria that produce the amylase enzyme to break down starch.
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This document discusses biochemical tests used to identify members of the Enterobacteriaceae family, including lactose fermentation, IMViC (indole, methyl red, Voges-Proskauer, and citrate) tests, urease test, motility test, and hydrogen sulfide production test. It provides details on the principles, methods, expected results and significance of each test for differentiating bacterial species like E. coli, Klebsiella, Proteus, and Salmonella. The document is authored by Dr. Nabil El Aila and focuses on diagnostic microbiology of gram-negative bacteria.
Biochemical reactions new
INDOLE TEST
UREASE TEST
CITRATE TEST
METHYL RED(MR) TEST
VOGES – PROSKAUER(VP) TEST
TRIPLE SUGAR IRON(TSI) TEST
OXIDASE TEST
CATALASE TEST
CATALASE TEST-Principle:-
This test demonstrates presence of catalase enzyme. This enzyme catalyses the release of O2 from H2O2.
catalase
2H2O2 H2O + increase O2
Reagents:- 1) 3% H2O2.
2) 24 hrs cultured organisms
Procedure:-
With sterile wooden stick transfer culture organisms to test tube containing 3% H2O2 and observe for production of effervescence.
It can also be tested directly on growth plate.
Positive Control: Staphylococci.
Negative Control: Streptococci.
False positive reactions:
If culture medium contains catalase enzyme e.g., blood agar, chocolate agar.
If iron wire loop is used
Bacterial identification
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2. The coagulase test identifies Staphylococcus aureus by observing if plasma clots due to coagulase production. S. aureus produces free and bound coagulase.
3. The oxidase test detects oxidase production in bacteria like Pseudomonas by observing the development of color when bacteria is added to oxidase reagent.
4. The indole test identifies if bacteria
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UREASE TEST
CITRATE TEST
METHYL RED(MR) TEST
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This test demonstrates presence of catalase enzyme. This enzyme catalyses the release of O2 from H2O2.
catalase
2H2O2 H2O + increase O2
Reagents:- 1) 3% H2O2.
2) 24 hrs cultured organisms
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With sterile wooden stick transfer culture organisms to test tube containing 3% H2O2 and observe for production of effervescence.
It can also be tested directly on growth plate.
Positive Control: Staphylococci.
Negative Control: Streptococci.
False positive reactions:
If culture medium contains catalase enzyme e.g., blood agar, chocolate agar.
If iron wire loop is used
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Catalase is an important enzyme that breaks down reactive oxygen species (ROS) such as hydrogen peroxide, protecting cells from oxidative damage. The catalase test detects this enzymatic activity by observing bubble formation when bacterial cultures or colonies are mixed with hydrogen peroxide. A positive result indicates catalase production, allowing distinction between bacterial species - for example, Staphylococcus is catalase-positive while Streptococcus is negative. The procedure involves placing a small amount of bacterial growth on a slide and mixing with hydrogen peroxide, with bubble formation signifying a positive catalase test result.
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3) Biochemical tests including IMViC (Indole, Methyl Red, Voges Proskauer, Citrate) which identify enteric bacteria based on fermentation patterns.
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- Important pathogenic genera include Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter, Citrobacter, and Yersinia. They can cause diseases like urinary tract infections, diarrhea, pneumonia, and sepsis.
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2. Methyl red test detects acid production in MR-VP broth, turning yellow for no acid and red for acid. Used to differentiate E. coli and E. aerogens.
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1. Enzymatic and metabolic pathway tests like carbohydrate fermentation and amino acid degradation.
2. Inhibitor profile tests using antibiotics like bacitracin, novobiocin, and vancomycin to differentiate bacterial species.
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1. Determining an isolate's nutritional and metabolic capabilities through a combination of enzyme and inhibitor tests is commonly used to identify its genus and species.
2. Tests establish enzymatic capabilities and ability to grow under stressors like salts, surfactants, toxins and antibiotics. Enzyme tests measure single enzymes or full pathways, while inhibitor tests examine susceptibility.
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This document provides information about an upcoming exam on protein purification techniques and related topics. It outlines the skills and concepts students should have learned, including using pipettes, spectrophotometers, organizing protocols, and preparing reports. It notes an upcoming protein purification lab experiment and encourages students to pay attention to details and properly label all samples to avoid issues. Finally, it provides an overview of the protein purification strategy and techniques to be used, including ammonium sulfate precipitation, dialysis, and column chromatography.
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Work Report- October 2021.pptx
The document reports on work to characterize genetic mutations that confer high-level beta-lactam resistance in Oxacillin-resistant mutants derived from Oxacillin-susceptible MRSA (OS-MRSA). It describes preparing competent cells from several mutant strains to conduct allelic exchange and complementation experiments by electroporating them with the pIMAY plasmid containing genes identified as mutated. The experiments aim to construct complementation strains of Oxacillin resistant OS-MRSA mutants to confirm the roles of identified mutations in conferring resistance.
Immobilisation cell culture
This document discusses cell immobilization techniques for cell culture. It describes three main types of cell culture: adherent cell culture, suspension cell culture, and immobilized cell culture. Immobilized cell culture involves encapsulating, adsorbing, or entrapping cells within a polymeric or porous matrix. The document then discusses types of cell immobilization including encapsulation, entrapment, adsorption, and cross-linking. Specific examples provided include immobilizing yeast cells in calcium alginate beads and using immobilized cells in bioreactors. Benefits of immobilized cell culture include higher cell densities, longer culture stability, and protection from shear forces.
Advanced Cell Culture Lecture 2 - CBNU
This document outlines the syllabus for an Advanced Animal Cell Culture course. It lists the topics that will be covered in each class, including introductions to cell culture, stem cells, transgenic and knockout animals, and genome engineering. It also provides details about some of the topics, such as commonly used cell lines and model organisms, cell culture techniques like subculture and cryopreservation, and research methods using cultured cells like microscopy, staining, and molecular biology techniques. The document emphasizes that cultured cells can serve as useful model systems to study specific cell types and biological phenomena while avoiding complications from whole organisms.
Chemistry of proteins
Proteins are the most abundant and functionally diverse molecules in living systems. They perform essential functions like structure, movement, transport, defense, and regulation of processes. Proteins are classified based on their chemical properties, functions, and nutritional importance. Some major classifications include structural, enzymatic, transport, and storage proteins. Proteins can be precipitated through various means like heat denaturation, dehydration using alcohol, increasing salt concentrations, and reactions with heavy metals or alkaloids. Precipitation tests are used to separate and identify proteins.
Bacteriology
This document discusses various biochemical and physiological tests that can be used to identify different groups of bacteria. It provides details on 14 common biochemical tests, including IMVIC (indole, citrate, motility, Voges-Proskauer), catalase production, urease, carbohydrate fermentation, and gelatin liquefaction. It also describes 7 physiological tests to characterize bacteria, such as examining their growth under different temperatures, pH levels, carbon and nitrogen sources, and salt concentrations. The document serves as a reference for the types of biochemical and physiological analyses that can be performed to classify and identify unknown bacterial species.
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An extensive coverage of Western Blotting including protein extraction, protein quantification, PAGE, transfer, staining and densitometry
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DIALISIYS
- 3. All enzymes are Proteins But all proteins are not enzymes Based on their nature Proteins are divided into two 1. Intra cellular 2. Extra cellular Partial Purification Of Protein abhi creations 3
- 4. Protein Separation Or Precipitation Ammonium Sulphar Precipitaion Ruputure of cell membrane (in case of intra celluar ) Strong acid / Alkali /SDS/Heat (SDS- Sodium Dodozyl Sulphate ) It is used in case of bacterial cell abhi creations 4
- 5. 4 molar Ammonium Sulphate needed for the pecipitation of Bacteria Concentration is based on the sathuration level of Ammonium Sulphate . Overnight incubation (at 4⁰C) abhi creations 5
- 6. Purification of Protein Selection of the protein K Breakage of cell membrane SPS Ammonium Sulphate Precipitation 4M at 4⁰C Dialysis abhi creations 6
- 8. Dialysis Selection of Dialysis membrane Molecular weight cutoff Eg:- 20-200, 200-400, 400-600, 600- above Dialysis membrane activation buffer Activation Hollow structure Membrane tie (Activated dialysis bag) abhi creations 8
- 9. Adding Ammonium sulphate to the bag Dialysis Against buffer (PBS) Ph - 7 abhi creations 9