Bmsc 210 Mid1 p2

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Scottygo
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Bmsc 210 Mid1 p2
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2013-01-29 16:34:42
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Bmsc 210 Mid1 p2
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  1. Bright-field scope
    - Specimens are visualized because of differences in contrast (density) between specimen and surroundings

    • Two sets of lenses form the image
    • -Objective lens and ocular lens
    • – Total magnification = objective magnification x ocular magnification– Maximum magnification is ~2,000x
  2. Resolution:
    the ability to distinguish two adjacent objects as separate and distinct
  3. – Resolution distance
    • -the smallest distance between two distinguishable objects
    • -proportional to wavelength of light used and inversly proportional to numerical aperture of lens
    • – Limit of resolution for light microscope is about 0.2 um
    • -blue light has short wavelenght (increases resolution) red has long (decreases resolution)
    • -high NA increases resolution
  4. 4 steps to preparing a smear
  5. Differential stains:
    • • Differential stains separate bacteria into groups
    • • The Gram stain is widely used in microbiology
  6. Gram-positive bacteria appear _____ After staining
    purple
  7. gram negative bacteria appear ___ after staining
    red
  8. 4 Steps to gram stain
  9. Phase-Contrast Microscopy
    • – Invented in 1936 by Frits Zernike
    • – Phase ring amplifies differences in the refractive index of cell and surroundings
    • – Improves the contrast of a sample without the useof a stain
    • – Allows for the visualization of live samples– Resulting image is dark cells on a light background
  10. Dark-Field Microscopy
    • – Light reaches the specimen from the sides
    • – Light reaching the lens has been scattered byspecimen
    • – Image appears light on a dark background
    • -Excellent for observing motility
  11. Fluorescence Microscopy
    • – Used to visualize specimens that fluoresce
    • • Emit light of one color when illuminated with another color of light
    • – Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI
    • – Widely used in microbial ecology for enumerating bacteria in natural samples
  12. Differential Interference Contrast (DIC)Microscopy
    • – Uses a polarizer to create two distinct beamsof polarized light
    • – Gives structures such as endospores,vacuoles, and granules a three
    • -dimensionalappearance
    • – Structures not visible using bright
    • -fieldmicroscopy are sometimes visible using DIC
  13. Atomic Force Microscopy (AFM)
    • – A tiny stylus is placed close to a specimen
    • – The stylus measures weak repulsive forcesbetween it and the specimen
    • – A computer generates an image based on thedata received from the stylus
  14. Confocal Scanning Laser Microscopy (CSLM)
    • – Uses a computerized microscope coupled with a laser source to generate a three
    • -dimensional image
    • – Computer can focus the laser on single layers of the specimen
    • – Different layers can then be compiled for a three dimensional image
    • – Resolution is 0.1 um for CSLM
  15. Electron microscopes use ____ insteadof photons to image cells and structures
    electrons
  16. • Transmission Electron Microscopy (TEM)
    •  
    • – Electromagnets function as lenses
    • – System operates in a vacuum
    • – High magnification and resolution (0.2 nm)– Enables visualization of structures at the molecular level
    • – Specimen must be very thin (20–60 nm) and be stained
  17. Scanning Electron Microscopy (SEM)
    • – Specimen is coated with a thin film of heavy metal(e.g., gold)
    • – An electron beam scans the object
    • – Scattered electrons are collected by a detectorand an image is produced
    • – Even very large specimens can be observed
    • – Magnification range of 15x–100,000x

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