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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 bacteria are enough!. Historical Microscopy. Anton van Leeuwenhoek-1670’s 1 st to see micro-organisms

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Microbiology chapter 3 microscopy and staining l.jpg

Microbiology Chapter 3Microscopy and Staining

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What’s 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-1670’s

  • 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 Light:Wavelength 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 Light:Light 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 Light:Light 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 eyepiece=1000X

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Light Microscopy and Types of Microscopes

  • Microscope that uses visible light to observe specimen

  • Hooke’s 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 (STM’s)-

    - 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 won’t take up stain

  • Flagellar staining- coats flagella so they can be seen

  • Endospore staining- Schaeffer-Fulton stain