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Chapter 10

Chapter 10. Classification of Microorganisms. The Study of Phylogenetic Relationships. Taxonomy The science of classifying organisms Provides universal names for organisms Provides a reference for identifying organisms Systematics, or Phylogeny

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Chapter 10

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  1. Chapter 10 Classification of Microorganisms

  2. The Study of Phylogenetic Relationships Taxonomy • The science of classifying organisms • Provides universal names for organisms • Provides a reference for identifying organisms Systematics, or Phylogeny • The study of the evolutionary history of organisms

  3. The Three-Domain System Genetic evidences Universal ancestor split into 3 lineages Figure 10.1

  4. Placing BacteriaAnton van Leeuwenhoek (1673) 1735 Kingdoms Plantae and Animalia 1857 Bacteria and fungi put in the Kingdom Plantae –“Flora” 1866 Kingdom Protista proposed for bacteria, protozoa, algae, and fungi 1937 Prokaryote introduced for cells "without a nucleus" 1961 Prokaryotedefined as cell in which nucleoplasm is not surrounded by a nuclear membrane 1959 Kingdom Fungi 1968 Kingdom Prokaryotae proposed 1978 Two types of prokaryotic cells found

  5. Table 10.1

  6. Fossilized Prokaryotes 3.5 billion years ago Figure 10.4a

  7. Fossilized Prokaryotes Figure 10.4b

  8. Fossilized Prokaryotes Figure 10.4c

  9. A Model of the Origin of Eukaryotes: Endosymbiotic Theory Cyanophora paradoxa (algae): Symbiosis between a bacterium and an Eukariotic cell (1978) Figures 10.2, 10.3

  10. Phylogenetics • Grouping organisms according to common properties implies that a group of organisms evolved from a common ancestor • Each species retains some characteristics of its ancestor • SEQUENCING ANALYSIS of Thermotoga maritima => gene homologues to Eubacteria and Archaebacteria => Branching point between two domains

  11. Phylogenetic Relationships of Prokaryotes Thermotoga maritima Figure 10.6

  12. IMPORTANT DATES • 4.5 BILLION-ORIGIN OF THE EARTH • 3.5 BILLION-PROKARYOTES DOMINATE NATURAL ENVIRONMENT Oldest photosynthetic bacterial fossil • 2.5 BILLION-O2 ACCUMULATES IN THE ATMOSPHERE • 1.5 BILLION-EUKARYOTES DOMINATE • 0.5 BILLION-CAMBRIAN EXPLOSION OF MULTICELLULAR EUKARYOTE ORGANISMS

  13. Scientific Nomenclature • Common names • Vary with languages • Vary with geography • Binomial Nomenclature (genus + specific epithet) • Used worldwide • Escherichia coli • Homo sapiens

  14. Scientific Names

  15. Taxonomic Hierarchy Domain Kingdom Phylum Class Order Family Genus Species

  16. The Taxonomic Hierarchy Figure 10.5

  17. Classification of Prokaryotes • Prokaryotic species: A population of cells with similar characteristics • Culture: Grown in laboratory media • Clone: Population of cells derived from a single cell • Strain: Genetically different cells within a clone

  18. Classification of Eukaryotes • Animalia: Multicellular; no cell walls; chemoheterotrophic • Plantae: Multicellular; cellulose cell walls; usually photoautotrophic • Fungi: Chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores or hyphal fragments • Protista: A catchall kingdom for eukaryotic organisms that do not fit other kingdoms • Grouped into clades based on rRNA

  19. Classification of Eukaryotes • Eukaryotic species: A group of closely related organisms that breed among themselves

  20. Classification of Viruses • Viral species: Population of viruses with similar characteristics that occupies a particular ecological niche

  21. Classification and Identification • Classification: Placing organisms in groups of related species. Lists of characteristics of known organisms. • Identification: Matching characteristics of an “unknown” organism to lists of known organisms. • Clinical lab identification

  22. References

  23. References

  24. A Clinical Microbiology Lab Report Form Figure 10.7

  25. Identification Methods • Morphological characteristics: Useful for identifying eukaryotes • Differential staining: Gram staining, acid-fast staining • Biochemical tests: Determines presence of bacterial enzymes • Provide insights into species niche in the ecosystem • » Diagnostic, epidemiological value Other: Rapid ID Methods (Multitest Systems: API, Enterotube)

  26. BIOCHEMICAL TESTS Catabolism Substrate Waste Products H2O Exoenzymes Energy Endoenzymes Cellular material Clinical diagnosis: Specific series of biochemical tests (+ Selective/differential media) have been develop for fast identification.

  27. BIOCHEMICAL DIFFERENTIAL TESTS • CATALASE TEST • Detects production of Catalase by microbes • H2O2 + H2O2 2H2O + O2 (gas) • H2O2 + XH2 2H2O + X • Substrate: 10 % solution of H2O2 • Performed directly on non-blood containing medium • Otherwise, performed on a clean slide Catalase Peroxidase

  28. BIOCHEMICAL DIFFERENTIAL TESTS • CATALASE TEST

  29. BIOCHEMICAL DIFFERENTIAL TESTS • CARBOHYDRATE FERMENTATION • pH indicator: Phenol Red • Acid production • Aerogenic fermenters • Anaerogenic fermenters Fermentation is the process of obtaining energy from organic compounds, using an organic molecule as a final electron acceptor (Not requiring Oxygen)

  30. BIOCHEMICAL DIFFERENTIAL TESTS • NITRATE REDUCTION • Nitrate Reductase • NO3- + 2 e- + 2 H+ —> NO2- + H2O • Nitrite Reductase • 2 NO2- + 10 e- + 12 H+ —> N2 + 6 H2O • Evidence for a multistep process • NO3- NO2- NO • NO N2O N2 Nitrate Reductase Nitrite Reductase Nitric Oxide Reductase Nitrous Oxide Reductase

  31. BIOCHEMICAL DIFFERENTIAL TESTS NITRATE REDUCTION Reagents: sulfanilic acid/-naphtylamine No color change Red color Bacterial Reduction NO3- NO2- Add powdered Zn No color change NO3- NO2- NH2/N2 Nitrate Reductase/Nitrite Reductase both present Red color no Bacterial Reduction a chemical reduction of NO3- NO2-

  32. BIOCHEMICAL DIFFERENTIAL TESTS • NITRATE REDUCTION

  33. BIOCHEMICAL DIFFERENTIAL TESTS • CITRATE UTILIZATION TEST • Basis: Utilization of citrate as sole source of carbon • Medium: Citrate Broth/Simmon’s Citrate Agar slant • Sodium citrate Acetate + Oxaloacetate • Citrate permease also required for entry • Indicator: Bromthymol blue • Accumulation of alkaline products lead to indicator color change • Positive: growth & change in color indicator to blue • Negative: no growth nor change in indicator color (remains green-acidic)

  34. CITRATE UTILIZATION TEST • RESULTS

  35. O O CH3—C—C—OH BIOCHEMICAL DIFFERENTIAL TESTS • INDOLE TEST • Detects intracellular enzyme complex Tryptophanase Medium: Tryptone Broth Tryptophanase NH3 Indole Pyruvic Acid Ammonia • Kovac’s Reagent (p-dimethylaminobenzaldehyde) • Added after 24 hr. incubation

  36. BIOCHEMICAL DIFFERENTIAL TESTS • INDOLE TEST RESULTS

  37. Hydrolytic & Degradative reactions • STARCH HYDROLYSIS • Urea Hydrolysis • Fat Hydrolysis

  38. RAPID IDENTIFICATION METHODS (Multitest Systems): ENTEROTUBE

  39. Numerical Identification Figure 10.9

  40. RAPID ID METHODS-API 20 E

  41. Dichotomous Key Figure 10.8

  42. Dichotomous Key

  43. Dichotomous Key ANIMATION Dichotomous Keys: Overview ANIMATION Dichotomous Keys: Practice

  44. Serology • Base on the specificity of antigen-antibody reaction • Combine known antiserum plus unknown bacterium • Slide agglutination test Figure 10.10

  45. ELISA • Enzyme-linked immunosorbent assay • Known antibodies placed in well of a microplate • Unknown type of bacterium is added • Antibodies linked to enzyme: color revealed • Enzyme substrate • Fast; scan and read by a computer • Many samples at a time Figure 18.14

  46. An ELISA Test Known antibodies placed in well Figure 10.11

  47. Indirect ELISA

  48. The Western Blot http://www.youtube.com/watch?v=iUrK9FuMAgs&list=PL3156C723DEA979C9 Figure 10.12

  49. Phage TypingTest to determine which phages a bacterium is susceptible to Phage Typing of Salmonella enterica Figure 10.13

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