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Microbiology Chapter 3 Microscopy and Staining

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Microbiology Chapter 3. Microscopy and Staining. What's on a Pinpoint? How many bacteria? ... How many are needed to start an infection? Sometimes as few as 10 ... – PowerPoint PPT presentation

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Title: Microbiology Chapter 3 Microscopy and Staining


1
Microbiology Chapter 3Microscopy and Staining
2
Whats on a Pinpoint?
  • How many bacteria?
  • How many are needed to start an infection?
  • Sometimes as few as 10 bacteria are enough!

3
Historical Microscopy
  • Anton van Leeuwenhoek-1670s
  • 1st to see micro-organisms
  • lens maker, simple scopes 100x to 300x
  • Single lens, like a magnifying glass
  • Studied animalcules

4
Principles of Microscopy
  • Metric units- powers of 10
  • Microscopy- technology of making very small
    things visible to naked eye
  • Measurements in
  • - micrometers (microns) um 0.000001m 10-6 m
  • - nanometers nm 10-9 m
  • - angstroms- (A) 10-10 m

5
Properties of LightWavelength and Resolution
  • Wavelength- length of a light ray
  • Resolution- ability to see 2 objects as separate
    discrete units (not fuzzy)
  • Visible light 550nm (NG)
  • UV light 100-400 nm better for resolution
  • Electron microscopy- .005 nm high reso
  • Resolving power of lens- numerical measure of
    lens, smaller distance from lens to slide
    greater resolving power

6
Properties of LightLight and Objects
  • Reflection-light strikes an object bounces
    back
  • Transmission- light passes through object
  • Absorption- light rays taken up by object
  • Luminescence-absorbed UV rays are changed to
    longer wave reemitted
  • Fluoresce- luminescence only occurring during
    irradiation
  • Phosphorescent- object emits light when light
    rays no longer strike it (some bacteria)

7
Properties of LightLight and Objects
  • Refraction- bending of light as it passes from
    one medium to another
  • Index of refraction- measure of the speed at
    which light penetrates
  • Immersion oil- used for better resolution because
    oil as the same index of refraction as glass.
  • Diffraction- light waves bend around an opening
    and could cause blurry slides
  • Iimit oil immersion with 10 x eyepiece1000X

8
Light Microscopy and Types of Microscopes
  • Microscope that uses visible light to observe
    specimen
  • Hookes compound microscope had more than 1
    lens
  • The Compound Light Microscope
  • - monocular- 1 eyepiece, binocular-2
  • Survey of microscope parts and their functions
    pg 58

9
Total Magnification Calculations
  • Scanning power -4x X 10x (ocular) 40x
  • Low power 10x X 10x(ocular) 100 x
  • High dry power 40x X 10 x 400 X
  • Oil immersion 100x X 10 x 1000x
  • Parfocal- in focus on one power, simple rotate
    nosepiece and its should focus on next power
  • Ocular micrometer- measure size of sample

10
Light Microscopy and Types of Microscopes
  • Dark-Field Microscopy- condenser causes light to
    reflect off specimen at an angle and increases
    the contrast
  • Phase-Contrast Microscopy-to observe live and
    unstained specimens by increasing refractive
    index and shows different degrees of brightness
  • Nomarski Microscopy- differential interference
    contrast and looks 3D

11
Light Microscopy and Types of Microscopes
  • Flourescence- UV light is used to excite
    molecules, longer wavelengths bright
  • Confocal Microscopy- usesbeams of UV lases light
    and computer reconstructs images, up to 40
    better. Can study microbes alive or not.
  • Digital Microscopy-have built in digital camera
    and can be viewed on screen

12
Different Types of Electron Microscopy
  • EM uses electron beam and electro-magnets not
    lenses- high resolution
  • Photos taken Electron micrographs
  • Transmission Electron Microscopy- (TEM) better
    view of internal structures up to 500,000x
    magnification
  • - shadow casting-
  • - freeze fracturing-
  • - freeze etching-

13
Different Types of Electron Microscopy
  • Scanning Election Microscopy (SEM)-
  • - Image of the surface 3D 50,000x mag
  • Scanning Tunneling Microscopy (STMs)-
  • - 1980 can be used with liver specimens and
    under water
  • Atomic force microscope-(AFM)- advanced 3d from
    atomic size to 1 micron
  • - used to study DNA, proteins

14
Techniques of Light Microscopy
  • Preparation of Specimens for the Light
    Microscope
  • 1) Wet Mounts- drop of medium with microbes is
    spread on a slide
  • 2) Smears- microbes from a loopful of medium are
    spread on a slide, then heat fixed to kill
    microbes
  • - heat fixation-

15
Principles of Staining
  • Stain- dye that binds to a cellular structure
    and gives it color
  • charge-basic methylene blue, crystal violet,
    safranin and malachite green
  • - charge-acidic eosin and picric acid
  • Simple stain- single dye and reveals basic cell
    shapes and structures
  • Differential stain- 2 or more dyes Gram stain,
    Ziehl-Neelsen acid fast and spore

16
Gram Stain
  • Gram Stain- 1884 crystal violet () and iodine
    and ethanol decolorizer, and counterstained with
    safranin (-)
  • Gram purple
  • Gram - red
  • Gram non reactive no stain
  • Gram Variable stain unevenly

17
Special Staining Procedures
  • Ziehl-Neelsen Acid-Fast Stain
  • - 1882 modification of Ehrlich staining method
  • - Acid fast retain red color in cell walls
  • Negative staining-capsule is present and wont
    take up stain
  • Flagellar staining- coats flagella so they can
    be seen
  • Endospore staining- Schaeffer-Fulton stain
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