MICR20010F.pptx

MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín Exams • Online Practical Exam – 2-3pm November 24th – 30 MCQ Questions over 1 hours – Only on Practical material (manual/online material) – Worth 15% of grade • In person Final Exam in RDS – 16.30-18.30pm Friday 15 th December – Confirm on...

MICR20010 Agricultural Microbiology Dr. Tadhg Ó Cróinín Exams • Online Practical Exam – 2-3pm November 24th – 30 MCQ Questions over 1 hours – Only on Practical material (manual/online material) – Worth 15% of grade • In person Final Exam in RDS – 16.30-18.30pm Friday 15 th December – Confirm on Exam timetable – 60 MCQ Questions over 2 hours – On Lecture Material – Worth 70% of grade • Sample questions have been provided, Please try these • Follow up e-mail early next week with final directions for online MCQ MICR20010 - remaining lectures • • • • • • • Lecture 11 – Microorganisms and Disease Lecture 12 – The Immune System Lecture 13 - Pathogenic Bacteria Lecture 14 – Pathogenic Fungi and Viruses Lecture 15 – Antibiotic Resistant Microorganisms Lecture 16 – Identification of Microorganisms Lecture 17 – Microbiology in the Food Industry – The Fungi • Lecture 18 – Microbiology in the Food Industry Fermentations • Lecture 19 – The Nitrogen Cycle Sequence of events from Isolation to Identification Isolation to Identification Step 1 – Isolation of Specimens • Isolate from the site of infection • Blood, urine, feces, sputum, pus etc. • Biopsies • Both the growth media and the Specimen • For correct isolation + identification it must be obtained correctly • Must not introduce m/os into the sample • Must not damage factors required for identification Anaerobic Growth Conditions • In particular for wound or blood infection – Clostridia • Microaerophilic jars for other pathogens Helicobacter pylori, Campylobacter Growth of Bacteria for identification Growth media + culture 1. General purpose media: • Supports the growth of most aerobic + facultative aerobic m/os 2. Enriched media: • Metabolically fastidious m/os • Specific nutrients enhances specific growth 3. Selective media: • Media that enhances the growth of certain m/os whilst retarding others 4. Differential media: • Allows identification of m/os based on their appearance on media Growth of Bacteria for identification Eosin methylene blue (EMB) • Selective and differential • Isolation of Gram negative m/o • Lactose + sucrose, no glucose – Acid causes Eosin  black – Methylene blue inhibits most Gram pos E.coli…..colonies black with greenish sheen Salmonella…translucent pink Escherichia coli Escherichia coli and Salmonella paratyphi Flower on 2 EMB plates Biochemical tests in media look for: • Ability to ferment sugars (pH indicator dye) Biochemical tests in media look for: • Gas production – In liquid it is collected in vials – In agar, the agar is pushed up Fig 24.7 a + b Biochemical tests in media look for: • Numerous tests have been developed – Analytical Profile index 21 tests for enterobacteriaceae + other nonfastidious Gram neg rods • 20 tubes with dehydrated substrates • Inoculate with bacteria…..observe reaction • 21st test= oxidase test (E. coli is oxidase negative Pseudomonas is oxidase positive) Media tests Immunological tests Immunological tests Example of an antibody Antigen-binding site Gamma heavy Chain IgG Antigen-Antibody interaction for diagnosis Specificity of antibody • The ability of Ig to recognise a single antigen • Don’t want cross reaction • Don’t want false positives Sensitivity of antibody • Lowest amount of antigen that can be detected • If possible a single antigen • Test employed influences the level of Ig needed Assay Sensitivity (mg antibody/ml) (smallest amount of Ig to give a reaction with antigen) Agglutination reaction Direct Passive 0.08 0.4 Radioimmuno assay (RIA) 0.0008-0.008 Enzyme-linked immunosorbent Assay (ELISA) Immunofluorescence 8.0 0.0008-0.008 positive Tests 1. Neutralisation test Tests 1. Neutralisation test • Igs neutralise biological activity of pathogens/toxins • Can occur in vitro + in vivo 1. Viral neutralisation • Certain Viruses cause a cytopathic effect on invasion of cells • To test: – Mix Virus containing serum with specific Ig – Introduce into cell culture – Examine for ability of Igs to inhibit cytopathic effect Infection Cytopathic effect Viral neutralisation Viral neutralisation VIRUS Attachment sites CELL Viral neutralisation VIRUS CELL Viral neutralisation Viral neutralisation Viral neutralisation Virus can’t attack cell Infection Cytopathic effect Viral neutralisation Tests 2. Viral hemagglutination test Tests 2. Viral hemagglutination test • • • Not all viruses have cytopathic effect on invasion of cells Surface proteins may clump Red blood cells (RBC) Igs against influenza- inhibit this Agglutinated Non agglutinated Fig18.6 Microbiology : an introduction Ed: Tortora/Funke/Case Tests 3. Fluorescent Igs Tests 3. Fluorescent Igs • Igs modified with fluorescent dyes • Detect antigen on cells – Rhodamine B…..fluoresces Red – Fluorescein isothiocyante…yellow-green • Cells with tagged Igs bound..emit fluorescent colour Tests Fluorescent Igs: Methods of labelling Ig • • Direct Indirect: – Ig to antigen is not labelled. – A secondary Ig is labelled Direct fluorescence Bacteria Direct fluorescence Bacteria – antibody is labelled with tag Direct fluorescence Bacteria – antibody is labelled with tag A. Immunoflourecence directly From a sample Immunoflourecence of cells That have been infected with the Virus in A Indirect fluorescence 1st antibody not labelled Indirect fluorescence 1st antibody not labelled Indirect fluorescence 1st antibody not labelled Indirect fluorescence 1st antibody not labelled 2nd antibody labelled Indirect fluorescence 1st antibody not labelled 2nd antibody labelled Tests 4. Enzyme Linked Immunosorbent assay (ELISA) Tests Enzyme Linked Immunosorbent assay (ELISA) • Enzyme label – Peroxidase – Alkaline phosphatase – Beta-galatosidase • Typically use polystyrene plates • Types: – Direct….detect antigens – Indirect…detect Igs Tests Enzyme Linked Immunosorbent assay (ELISA) • Steps (detect Igs-indirect) 1. Wells coated with antigen 2. Unbound plastic blocked with non reactive protein (gelatin) 3. Sera sample added..Igs if present bind to antigens 4. Ig labelled is added 5. Enzyme substrate added….colour change Indirect… detect Igs Direct…. detect antigens PCR based tests • The Polymerase Chain Reaction • DNA replication in a tube • By designing primers to a specific region of DNA we can amplify a very specific product. • LAMP – PCR allows for carrying out of the reaction at a constant temperature. PCR Video Other Technologies • MALDI-TOF Mass spectrometry in clinical labs. • Different bacteria give different signature profiles and so can identify pure cultures. Next Monday on MICR20010 Microbiology in the Food Industry Dr. Tadhg Ó Cróinín