Microbiology Chapter 3 Microscopy and Staining

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Microbiology Chapter 3Microscopy and Staining
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Whats on a Pinpoint?

• How many bacteria?
• How many are needed to start an infection?
• Sometimes as few as 10 bacteria are enough!

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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-

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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

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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-

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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

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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

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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