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The Study of Microbial
Stru...
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Scale
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Discovery of
Microorganisms...
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Lenses and the Bending of
L...
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Lenses
• focus light rays a...
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Figure 2.2
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The Light Microscope
• many...
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The Bright-Field
Microscope...
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Figure 2.3
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Figure 2.4
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Microscope Resolution
• ab...
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•working distance
— distan...
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Figure 2.5
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Figure 2.6
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The Dark-Field Microscope
...
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Figure 2.7b
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The Phase-Contrast
Microsc...
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Figure 2.9
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Figure 2.10
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The Differential
Interfere...
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The Fluorescence
Microscop...
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Figure 2.12
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Figure 2.13c and d
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Preparation and Staining o...
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Fixation
• process by whic...
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Dyes and Simple Staining
•...
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Dyes and Simple Staining
•...
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Differential Staining
• di...
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Gram staining
• most widel...
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Figure 2.14
primary
stain
...
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Figure 2.15c Escherichia c...
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Acid-fast staining
• parti...
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Staining Specific
Structur...
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Staining Specific
Structur...
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Electron Microscopy
• beam...
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The Transmission Electron
...
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Figure 2.23
EM
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Specimen Preparation
• ana...
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Other preparation methods
...
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Figure 2.25
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Ebola
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Fly head
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The Scanning Electron
Micr...
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Figure 2.27
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Newer Techniques in
Micros...
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Confocal Microscopy
• conf...
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Figure 2.29
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Figure 2.30
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Scanning Probe
Microscopy
...
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Scanning Probe
Microscopy
...
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  • Ebola
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  • Microscope

    1. 1. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 The Study of Microbial Structure: Microscopy and Specimen Preparation
    2. 2. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 Scale
    3. 3. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3 Discovery of Microorganisms • Antony van Leeuwenhoek (1632-1723) – first person to observe and describe micro- organisms accurately Figure 1.1b
    4. 4. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 4 Lenses and the Bending of Light • light is refracted (bent) when passing from one medium to another • refractive index – a measure of how greatly a substance slows the velocity of light • direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface
    5. 5. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5 Lenses • focus light rays at a specific place called the focal point • distance between center of lens and focal point is the focal length • strength of lens related to focal length – short focal length ⇒more magnification
    6. 6. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 Figure 2.2
    7. 7. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 The Light Microscope • many types – bright-field microscope – dark-field microscope – phase-contrast microscope – fluorescence microscopes • are compound microscopes – image formed by action of ≥2 lenses
    8. 8. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 8 The Bright-Field Microscope • produces a dark image against a brighter background • has several objective lenses – parfocal microscopes remain in focus when objectives are changed • total magnification – product of the magnifications of the ocular lens and the objective lens
    9. 9. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 9 Figure 2.3
    10. 10. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 10 Figure 2.4
    11. 11. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 11 Microscope Resolution • ability of a lens to separate or distinguish small objects that are close together • wavelength of light used is major factor in resolution shorter wavelength ⇒ greater resolution
    12. 12. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12 •working distance — distance between the front surface of lens and surface of cover glass or specimen
    13. 13. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 13 Figure 2.5
    14. 14. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 14 Figure 2.6
    15. 15. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 15 The Dark-Field Microscope • produces a bright image of the object against a dark background • used to observe living, unstained preparations
    16. 16. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 16 Figure 2.7b
    17. 17. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 17 The Phase-Contrast Microscope • enhances the contrast between intracellular structures having slight differences in refractive index • excellent way to observe living cells
    18. 18. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18 Figure 2.9
    19. 19. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19 Figure 2.10
    20. 20. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 20 The Differential Interference Contrast Microscope • creates image by detecting differences in refractive indices and thickness of different parts of specimen • excellent way to observe living cells
    21. 21. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 21 The Fluorescence Microscope • exposes specimen to ultraviolet, violet, or blue light • specimens usually stained with fluorochromes • shows a bright image of the object resulting from the fluorescent light emitted by the specimen
    22. 22. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 22 Figure 2.12
    23. 23. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 23 Figure 2.13c and d
    24. 24. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 24 Preparation and Staining of Specimens • increases visibility of specimen • accentuates specific morphological features • preserves specimens
    25. 25. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 25 Fixation • process by which internal and external structures are preserved and fixed in position • process by which organism is killed and firmly attached to microscope slide – heat fixing • preserves overall morphology but not internal structures – chemical fixing • protects fine cellular substructure and morphology of larger, more delicate organisms
    26. 26. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 26 Dyes and Simple Staining • dyes – make internal and external structures of cell more visible by increasing contrast with background – have two common features • chromophore groups – chemical groups with conjugated double bonds – give dye its color • ability to bind cells
    27. 27. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 27 Dyes and Simple Staining • simple staining – a single staining agent is used – basic dyes are frequently used • dyes with positive charges • e.g., crystal violet
    28. 28. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 28 Differential Staining • divides microorganisms into groups based on their staining properties – e.g., Gram stain – e.g., acid-fast stain
    29. 29. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 29 Gram staining • most widely used differential staining procedure • divides Bacteria into two groups based on differences in cell wall structure
    30. 30. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 30 Figure 2.14 primary stain mordant counterstain decolorization positive negative
    31. 31. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 31 Figure 2.15c Escherichia coli – a gram-negative rod
    32. 32. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 32 Acid-fast staining • particularly useful for staining members of the genus Mycobacterium e.g., Mycobacterium tuberculosis – causes tuberculosis e.g., Mycobacterium leprae – causes leprosy – high lipid content in cell walls is responsible for their staining characteristics
    33. 33. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 33 Staining Specific Structures • Negative staining – often used to visualize capsules surrounding bacteria – capsules are colorless against a stained background
    34. 34. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 34 Staining Specific Structures • Spore staining – double staining technique – bacterial endospore is one color and vegetative cell is a different color • Flagella staining – mordant applied to increase thickness of flagella
    35. 35. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 35 Electron Microscopy • beams of electrons are used to produce images • wavelength of electron beam is much shorter than light, resulting in much higher resolution Figure 2.20
    36. 36. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 36 The Transmission Electron Microscope • electrons scatter when they pass through thin sections of a specimen • transmitted electrons (those that do not scatter) are used to produce image • denser regions in specimen, scatter more electrons and appear darker
    37. 37. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 37 Figure 2.23 EM
    38. 38. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 38 Specimen Preparation • analogous to procedures used for light microscopy • for transmission electron microscopy, specimens must be cut very thin • specimens are chemically fixed and stained with electron dense material
    39. 39. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 39 Other preparation methods • shadowing – coating specimen with a thin film of a heavy metal • freeze-etching – freeze specimen then fracture along lines of greatest weakness (e.g., membranes)
    40. 40. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 40 Figure 2.25
    41. 41. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 41 Ebola
    42. 42. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 42 Fly head
    43. 43. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 43 The Scanning Electron Microscope • uses electrons reflected from the surface of a specimen to create image • produces a 3-dimensional image of specimen’s surface features
    44. 44. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 44 Figure 2.27
    45. 45. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 45 Newer Techniques in Microscopy • confocal microscopy and scanning probe microscopy • have extremely high resolution • can be used to observe individual atoms Figure 2.20
    46. 46. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 46 Confocal Microscopy • confocal scanning laser microscope • laser beam used to illuminate spots on specimen • computer compiles images created from each point to generate a 3- dimensional image
    47. 47. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 47 Figure 2.29
    48. 48. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 48 Figure 2.30
    49. 49. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 49 Scanning Probe Microscopy • scanning tunneling microscope – steady current (tunneling current) maintained between microscope probe and specimen – up and down movement of probe as it maintains current is detected and used to create image of surface of specimen
    50. 50. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 50 Scanning Probe Microscopy • atomic force microscope – sharp probe moves over surface of specimen at constant distance – up and down movement of probe as it maintains constant distance is detected and used to create image
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