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33 Cards in this Set

  • Front
  • Back
What are 11 parts of a microscope?
-ocular lens
-objective lenses
-course focusing knob
-fine focusing knob
What type of microscopes can microbes be seen with?
What are the three important tasks a microscope must be able to do?
-magnification; enlarge specimen
-resolution; separate the details
-contrast; make details visible
What wavelength of light is best used for resolution?
-lower (shorter) wavelengths; towards blue and purple, x-rays, and gamma side of spectrum
When does magnification occur?
How does this occur in light and electron microscopy?
-magnification occurs when light is refracted as it passes through a lens
-light: uses glass lens
-electron: magnetic fields
What is the upper limit for light microscopy?
Why is this?
-resolution limits; beyond 2000x is empty magnification
What two functions contribute to resolution limits?
-wavelength of light
-numerical aperature
What is the formula for numerical aperature (NA)?
n= refractive index of material
sinQ= angular aperature of the objective
What are the refractive indexes of air, glass, and oil?
glass= 1.5
oil= 15.
What is the formula for resolution?
=.61 (wavelength)(NA)
*micrometers are units
What are some ways to increase resolution?
-lower wavelength
-increase NA (oil immersion)
-increase contrast (light differences, staining, or phasing light)
What are the different types of Light microscopy?
-bright-field microscopy
-dark-field microscopy
-differential interference contrast
How do phase microscopes work?
-treat light rays different from one another
-in phase light rays produce brighter image
-out of phase light rays produce darker images
-contrast created becuase light waves are 1/2 wavelength out of phase
How does fluorescent microscopy work?
-UV light is absorbed by specimen and energy is emitted as a longer, visible wavelength, AND this increases resolution and contrast
-some cells may beed staining or tagging (is green fluorescent protein)
What are 4 fluorescent staining proteins used in this type of microscopy?
-DAPI= blue (stains DNA)
-FITC= yellow/green
-rhodamine= red
-GFP= green
How does Laser-Scanning Confocal Microscopy work?
-UV lasers illuminate fluorescent chemicals in a single focal plane (no thicker than 1um)
-This light passes through pinhole aperture increasing resolution up to 40%
-Computer constructs 3-D image from digitized images of multiple optical slices
What are the two types of electron microscopy?
-transmission electron microscopes
-scanning electron microscopes
How close can light microscopes get to object and still resolve it? Why not closer?
-no closer than 200nm becuase the shortest wavelength of visible light is 400nm
Why do electron microscopes have greater resolving power and magnification?
-because electrons have wavelengths of .01-.001nm
How much can an electron microscope magnify an object?
How are images seen (especially if clear object)?
-Use heavy metals to stain structures or backgrounds
How does a transmission electron microscope work?
-electrons pass through specimen then magnetic field that focuses them onto fluorescent screen (which absorbs electrons)
What are the stipulations (limiting factors) to using a TEM (transmission Electron Microscope)?
How to absorbed e- show up?
-must be done with dead samples in a vacuum (air absorbs electrons)
-must be thin slices to allow electrons to pass through (~100nm)
-dark areas result from e- absorption
How does a scanning electron microscope work?
-sample is coated with metal and primary e- knock e- off metal producing secondary e- that are detected
-sectioning is not required (no absorption) but can only see outside of cells
What are the two types of probe microscopy?
-scanning tunneling microscopy
-atomic force microscopy
How does scanning tunneling microscopy work?
-uses metallic probe sharpened to end in a single atom
-measures flow of electrons (tunneling current) between probe and specimen's surface (must be conductive)
-can measure distances as small as 0.01 nm
How does atomic force microscopy work?
-same metallic probe
-tip of probe traverses the surface of specimen while a laser beam reflects off tip
-generates an atomic topography giving the same resolution as electron microscopy
*can use living specimens as no vacuum in needed
What are microbial stains composed of?
-salts (composed of cation and anion and one is colored: chromophore)
What is staining used for?
What are the three different types of staining covered in this class?
*to increase contrast and resolution
What type of stains are used most often?
-acidic and basic dyes:
methylene blue
cyrtal violet
What are three categories of differential staining?
-gram stains
-acid-fast stain
-endospore stain
What are two types of special stains?
-negative (capsule) stain
-flagellar stain
What are 3 Gram (+)bacteria seen in lab?
How about Gram (-)?
-staphylococcus, streptococcus, lactobacillus (many human pathogens and food microbes)
-E coli, Salmonella typhimurium (most widely studied bacteria; human pathogens)
What is the procedure for Gram staining?
-flood area with crystal violet for 1 minute, rinse with water
-flood with iodine for 1 min, rinse with water
-rinse with alcohol and acetone for 10-30 sec, rinse with water again
-flood with safranin for 1 minute (2 in lab), rinse with water, blot dry