Start here_ch04_lecture


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Start here_ch04_lecture

  1. 1. Chapter 4 Microscopy, Staining, and Classification
  2. 2. Units of Measurement Animation: Microscopy and Staining
  3. 3. Units of Measurement [INSERT TABLE 4.1]
  4. 4. Microscopy <ul><li>General Principles of Microscopy </li></ul><ul><ul><li>Wavelength of radiation </li></ul></ul><ul><ul><li>Magnification </li></ul></ul><ul><ul><li>Resolution </li></ul></ul><ul><ul><li>Contrast </li></ul></ul>
  5. 5. Microscopy [INSERT FIGURE 4.1]
  6. 6. Microscopy [INSERT FIGURE 4.2]
  7. 7. Microscopy [INSERT FIGURE 4.3]
  8. 8. Microscopy <ul><li>General Principles of Microscopy </li></ul><ul><ul><li>Contrast </li></ul></ul><ul><ul><ul><li>Differences in intensity between two objects, or between an object and background </li></ul></ul></ul><ul><ul><ul><li>Important in determining resolution </li></ul></ul></ul><ul><ul><ul><li>Staining increases contrast </li></ul></ul></ul><ul><ul><ul><li>Use of light that is in phase increases contrast </li></ul></ul></ul>
  9. 9. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Bright-field microscopes </li></ul></ul><ul><ul><ul><li>Simple </li></ul></ul></ul><ul><ul><ul><ul><li>Contain a single magnifying lens </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Similar to magnifying glass </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Leeuwenhoek used simple microscope to observe microorganisms </li></ul></ul></ul></ul>
  10. 10. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Bright-field microscopes </li></ul></ul><ul><ul><ul><li>Compound </li></ul></ul></ul><ul><ul><ul><ul><li>Use a series of lenses for magnification </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Light rays pass through specimen and into objective lens (one of a series of objective lenses) </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Oil immersion lens increases resolution because light does not refract </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Have one or two ocular lenses </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Total magnification = magnification of objective lens X magnification of ocular lens </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Most have condenser lens to direct light through specimen </li></ul></ul></ul></ul>
  11. 11. Microscopy [INSERT FIGURE 4.4]
  12. 12. Microscopy [INSERT FIGURE 4.5]
  13. 13. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Dark-field microscopes </li></ul></ul><ul><ul><ul><li>Best for observing pale objects </li></ul></ul></ul><ul><ul><ul><li>Only light rays scattered by specimen enter objective lens </li></ul></ul></ul><ul><ul><ul><li>Specimen appears light against dark background </li></ul></ul></ul><ul><ul><ul><li>Increases contrast and enables observation of more details </li></ul></ul></ul>
  14. 14. Microscopy [INSERT FIGURE 4.6]
  15. 15. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Phase microscopes </li></ul></ul><ul><ul><ul><li>Used to examine living organisms or specimens that would be damaged or altered by attaching them to slides or staining them </li></ul></ul></ul><ul><ul><ul><li>Treat one set of light rays differently from another set </li></ul></ul></ul><ul><ul><ul><li>Light rays in phase produce brighter image, while light rays out of phase produce darker image </li></ul></ul></ul><ul><ul><ul><li>Contrast is created because light waves are 1/2 wavelength out of phase </li></ul></ul></ul><ul><ul><ul><li>Two types </li></ul></ul></ul><ul><ul><ul><ul><li>Phase-contrast microscope </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Differential interference contrast microscope </li></ul></ul></ul></ul>
  16. 16. Microscopy [INSERT FIGURE 4.7]
  17. 17. Microscopy [INSERT FIGURE 4.8]
  18. 18. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Fluorescent microscopes </li></ul></ul><ul><ul><ul><li>Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength </li></ul></ul></ul><ul><ul><ul><li>UV light increases resolution and contrast </li></ul></ul></ul><ul><ul><ul><li>Some cells and molecules are naturally fluorescent, while others must be stained </li></ul></ul></ul><ul><ul><ul><li>Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins </li></ul></ul></ul>
  19. 19. Microscopy [INSERT FIGURE 4.9]
  20. 20. Microscopy [INSERT FIGURE 4.10]
  21. 21. Microscopy <ul><li>Light Microscopy </li></ul><ul><ul><li>Confocal microscopes </li></ul></ul><ul><ul><ul><li>Use fluorescent dyes </li></ul></ul></ul><ul><ul><ul><li>Use UV lasers to illuminate fluorescent chemicals in a single plane that is no thicker than 1.0  m </li></ul></ul></ul><ul><ul><ul><li>Resolution increased by up to 40% because emitted light passes through pinhole aperture </li></ul></ul></ul><ul><ul><ul><li>Computer constructs 3-D image from digitized images </li></ul></ul></ul>
  22. 22. Microscopy Animation: Light Microscopy
  23. 23. Microscopy <ul><li>Electron Microscopy </li></ul><ul><ul><li>Light microscopes cannot resolve structures closer than 200 nm because shortest wavelength of visible light is 400 nm </li></ul></ul><ul><ul><li>Electrons have wavelengths of 0.01 nm to 0.001 nm, so electron microscopes have greater resolving power and greater magnification </li></ul></ul><ul><ul><li>Magnifies objects 10,000X to 100,000X </li></ul></ul><ul><ul><li>Gives detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms </li></ul></ul><ul><ul><li>Two types </li></ul></ul><ul><ul><ul><li>Transmission electron microscopes </li></ul></ul></ul><ul><ul><ul><li>Scanning electron microscopes </li></ul></ul></ul>
  24. 24. Microscopy [INSERT FIGURE 4.11]
  25. 25. Microscopy [INSERT FIGURE 4.12]
  26. 26. Microscopy [INSERT FIGURE 4.13]
  27. 27. Microscopy Animation: Electron Microscopy
  28. 28. Microscopy <ul><li>Probe Microscopy </li></ul><ul><ul><li>Uses minuscule, pointed, electronic probes to magnify more than 100,000,000 times </li></ul></ul><ul><ul><li>Two types </li></ul></ul><ul><ul><ul><li>Scanning tunneling microscopes </li></ul></ul></ul><ul><ul><ul><li>Atomic force microscopes </li></ul></ul></ul>
  29. 29. Microscopy [INSERT FIGURE 4.14]
  30. 30. Microscopy [INSERT TABLE 4.2]
  31. 31. Staining <ul><li>Increases contrast and resolution by coloring specimens with stains/dyes </li></ul><ul><li>Smear of microorganisms (thin film) air dried to slide and then fixed to surface by heat or chemical fixation </li></ul><ul><li>Microbiological stains usually salts composed of cation and anion, with one colored (chromophore) </li></ul><ul><li>Acidic dyes stain alkaline structures; more commonly, basic dyes stain acidic structures </li></ul>
  32. 32. Staining [INSERT FIGURE 4.15]
  33. 33. Staining <ul><li>Simple stains </li></ul><ul><li>Differential stains </li></ul><ul><ul><li>Gram stain </li></ul></ul><ul><ul><li>Acid-fast stain </li></ul></ul><ul><ul><li>Endospore stain </li></ul></ul><ul><li>Special stains </li></ul><ul><ul><li>Negative (capsule) stain </li></ul></ul><ul><ul><li>Flagellar stain </li></ul></ul>
  34. 34. Staining [INSERT FIGURE 4.16]
  35. 35. Staining [INSERT FIGURE 4.17]
  36. 36. Staining [INSERT FIGURE 4.18]
  37. 37. Staining [INSERT FIGURE 4.19]
  38. 38. Staining [INSERT FIGURE 4.20]
  39. 39. Staining [INSERT FIGURE 4.21]
  40. 40. Staining [INSERT TABLE 4.3]
  41. 41. Staining <ul><li>Staining for Electron Microscopy </li></ul><ul><ul><li>Chemicals containing heavy metals used for transmission electron microscopy </li></ul></ul><ul><ul><li>Stains may bind molecules in specimens or the background </li></ul></ul>
  42. 42. Staining Animation: Staining
  43. 43. Classification & Identification of Microorganisms <ul><li>Taxonomy consists of classification, nomenclature, and identification </li></ul><ul><li>Enables scientists to organize large amounts of information about organisms and make predictions based on knowledge of similar organisms </li></ul>
  44. 44. Classification & Identification of Microorganisms <ul><li>Linnaeus, Whittaker, and Taxonomic Categories </li></ul><ul><ul><li>Linnaeus </li></ul></ul><ul><ul><ul><li>Provided system that standardized the naming and classification of organisms based on characteristics in common </li></ul></ul></ul><ul><ul><ul><li>Grouped similar organisms that can successfully interbreed into categories called species </li></ul></ul></ul><ul><ul><ul><li>Used binomial nomenclature in his system </li></ul></ul></ul>
  45. 45. Classification & Identification of Microorganisms
  46. 46. Classification & Identification of Microorganisms <ul><li>Linnaeus, Whittaker, and Taxonomic Categories </li></ul><ul><ul><li>Whittaker </li></ul></ul><ul><ul><ul><li>Linnaeus proposed only two kingdoms </li></ul></ul></ul><ul><ul><ul><li>Whittaker proposed a widely accepted taxonomic approach based on five kingdoms: Animalia, Plantae, Fungi, Protista, and Prokaryotae </li></ul></ul></ul>
  47. 47. Classification & Identification of Microorganisms [INSERT FIGURE 4.23]
  48. 48. Classification & Identification of Microorganisms <ul><li>Linnaeus, Whittaker, and Taxonomic Categories </li></ul><ul><ul><li>Linnaeus’s goal was classifying and naming organisms as a means of cataloging them </li></ul></ul><ul><ul><li>More modern goal is understanding relationships among groups of organisms </li></ul></ul><ul><ul><li>Major goal of modern taxonomy is to reflect phylogenetic hierarchy </li></ul></ul><ul><ul><li>Greater emphasis on comparisons of organisms’ genetic material led to proposal to add domain </li></ul></ul>
  49. 49. Classification & Identification of Microorganisms <ul><li>Domains </li></ul><ul><ul><li>Carl Woese compared nucleotide sequences of rRNA subunits (changes occur rarely) </li></ul></ul><ul><ul><li>Proposal of three domains based on three basic types of cells as determined by ribosomal nucleotide sequences </li></ul></ul><ul><ul><li>Three Domains: Eukarya, Bacteria, and Archaea </li></ul></ul><ul><ul><li>Cells in the three domains also differ with respect to many other characteristics </li></ul></ul>
  50. 50. Classification & Identification of Microorganisms <ul><li>Taxonomic and Identifying Characteristics </li></ul><ul><ul><li>Physical characteristics </li></ul></ul><ul><ul><li>Biochemical tests </li></ul></ul><ul><ul><li>Serological tests </li></ul></ul><ul><ul><li>Phage typing </li></ul></ul><ul><ul><li>Analysis of nucleic acids </li></ul></ul>
  51. 51. Classification & Identification of Microorganisms [INSERT FIGURE 4.24]
  52. 52. Classification & Identification of Microorganisms [INSERT FIGURE 4.25]
  53. 53. Classification & Identification of Microorganisms [INSERT FIGURE 4.26]
  54. 54. Classification & Identification of Microorganisms [INSERT FIGURE 4.27]
  55. 55. Classification & Identification of Microorganisms <ul><li>Taxonomic Keys </li></ul><ul><ul><li>Dichotomous keys – series of paired statements worded so that only one of two “either/or” choices applies to any particular organism </li></ul></ul><ul><ul><li>Key directs user to another pair of statements, or provides name of organism </li></ul></ul>
  56. 56. Classification & Identification of Microorganisms [INSERT FIGURE 4.28]
  57. 57. Classification & Identification of Microorganisms Animation: Dichotomous Keys