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Start here_ch04_lecture

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