Microscopy

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Microscopy

• Labs 1, 2 and 3

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Applications of Light Microscopy

• Observe less detailed features of intact cell
  than advanced microscopy (i.e. electron and
  laser)
• Shape, presence of flagella, diagnostic stain,
  arrangement of cells, some large internal
  features
• Brightfield
• Stained cells or cells with color/contrast
• External features
• Phase contrast
• Live transparent (unstained) cells
• Some internal features
• Dark-field
• Live transparent cells
• Greater resolution more features internal and
  external

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Path of Light Brightfield

• Illuminator
• Filter for shorter ? blue light
• Condenser
• Focuses light into specimen
• Digaphragm (iris)
• Controls amount of light to specimen
• Specimen on slide
• Diffracts light as it passes through?image
  produced
• Objective lenses at nosepiece
• Magnifies image 10, 40, or 100 times
• Image inverted
• Head with prism
• Direct light path into ocular lens
• Ocular lens magnifies image 10X
• Image path sent to eye
• Diopter is used to focus that image at correct
  focal length for eye.

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Magnification Versus Resolution

• Magnificationincrease in apparent size
• Objective and ocular lenses
• Resolutionclarity
• ability to see 2 nearby objects as distinct
  objects
• Resolving power depends on numerical aperture of
  lens
• Ability to gather light increase aperture and
  increase resolution of lens
• Increase magnification must increase aperture
  aperture limit
• Limit to mag with light microscope?1500X and
  resolution is bad at 1500X
• Highest resolution possible is 0.2µm with 100X
  lens
• Steps you can take to increase resolution
• GET MORE LIGHT TO LENS!
• oil immersion with 100X lens
• Same refractive index as glass prevents loss of
  light to air
• small wavelength light (blue light)
• Adjust diaphragm and condenser as you increase
  magnification
• Course focus and fine focus to focus image
• Clean lenses and slide

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Other stuff.

• Field of vision
• Smaller at higher powers must CENTER object or
  you will lose it at higher power
• Mechanical stage adjuster!!!
• Parfocal
• Focus under low power then move to high power
  immediately dont move focus in between then
  fine focus after you get to high power.
• Contrast versus resolution.
• you need light to increase resolution, but too
  much light can decrease contrast must find a
  happy medium.
• Spherical aberration
• Distortion of edges of field of view due to shape
  of lens.
• Look in the center, dude!
• Chromatic aberration
• Prism effect.
• We use achromatic lensesnew scopewe need not
  worry!!!!

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Cellular Morphology

• Prokaryotes
• Rods (staph, strep or single arrangement)
• Cocci (staph, strep or single arrangement)
• Spirillum
• No nuclei present.
• Must have cells under oil immersion to see any
  detail.
• Eukaryotes
• Cells much larger than prokaryotes
• Cant always see nucleus
• Fungi
• Molds versus single celled fungi
• Yeast
• Candida albicans vaginal yeast infection and
  thrush
• Saccharomyces cerevisiae bakers yeast brewing
  yeast

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Cellular Morphology Continued

• More Eukaryotes
• Fungi
• Molds
• Filamentous due to hyphae multicellular
• Penicillium
• Yes it makes penicillin grows on bread
• Aspergillus,
• Some produce aflotoxins (pathogenic)
• Rhizopus
• Classic bread mold and house mold
• Algae
• Have pigments associated with them single cell
• Dinoflagellate called Peridinium
• Protozoa
• Single cell
• Trypanosoma gambiensae
• Causes Sleeping Sickness nervous disorders
  central Africa tsetse flies
• Helminth
• Multi cell

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What to Do on 8/25-27

• Use directions on page 4-5 to look at the
  following slides
• a.      Schistosoma mansonni (40 X)
• b.      Peridinium (40X)
• c.      Trypanosoma gambiensae (mixed with RBCs
  look under oil immersion)
• d.      Penicillium (Mold types slide purple
  40X)
• e.      Rhizopus (Mold types slide pink 40X)
• f.        Aspergillus (Mold types slide green
  40X)
• g.      Yeast (40X and 100X to see nucleus)

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Phase Contrast

• Viewing live specimens
• Transparent brightfield light passes straight
  through
• Phase condenser diffracts light out of phase
  increases contrast of transparent objects
• Brownian movement versus motility
• Wet mount

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Yeast Brightfield
Yeast Phase Contrast
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Microbes in the Environment

• Where do microbes live and where do they not
  live?
• Sterile media versus culture.
• Autoclave
• To inoculate is to purposely grow an organism by
  putting it in some media, and to contaminate to
  to accidentally grow an unwanted organism.
• Broth versus solid media
• Agar
• Slant versus plate media
• Incubation

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Growth

• Growth in broth?cloudy or turbid
• Pellicle, sediment, flocculant
• Growth on plates?colonies
• Colony and colonial morphology
• Margin, elevation, color and colony shape
• Page 52 for terms to use
• Observe with dissecting scope
• Dissecting scopes used to observe 3D structure of
  larger objects while microscopes used to see 2D
  surface of smaller objects.

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What to Do on 9/3-8

• Directions page 12-14
• Modifications
• Do wet mount only
• Use pond water instead of hay infusion in 1.
• Repeat procedure for BM culture wet mount.
• Fill out lab report.
• Directions page 51-52
• Modifications
• Skip A and B
• Get 2 nutrient agar plates and 1 nutrient broth
  and do C
• Fill out lab report in book.

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Aseptic Technique

• Deep
• Slant
• Broth
• Sterile
• Asepsis
• Inoculate
• Demo the technique
• Describing your cultures
• Motile versus non-motile
• Slant descriptions
• Broth descriptions
• Can you tell if a broth is contaminated? How?

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What to do 9/8-10

• Pages 58-60
• Show modifications
• Inoculate one Deep (directions 4) one slant (3)
  and one broth (2) with PV
• Then inoculate a second deep, slant and broth
  with SA
• You should have a total of 6 tubes 2 deeps, 2
  slants and 2 broths.