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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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what s on a pinpoint
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
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”
principles of microscopy
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

properties of light wavelength and resolution
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
properties of light light and objects
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)
properties of light light and objects7
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
light microscopy and types of microscopes
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
total magnification calculations
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
light microscopy and types of microscopes10
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”
light microscopy and types of microscopes11
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
different types of electron microscopy
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-

different types of electron microscopy13
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

techniques of light microscopy
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-

principles of staining
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
gram stain
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
special staining procedures
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