Block One Lecture 3

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Author:
DesLee26
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282357
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Block One Lecture 3
Updated:
2014-09-07 22:45:16
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Garcia
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Microbiology
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September 7, 2014
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  1. Which measurements do we use?
    microns (1 micrometer= 10^-6 m=10^-3 mm)
  2. The Instruments


    A simple microscope has __.

    There is also the __.

    What is magnification?

    What are the magnifications in terms of lens?
    only one lens

    binocular compound microscope

    magnification= optical lens x objective

    4X scanning lens

    10X low power

    40X high power
  3. What is the resolution?

    To describe structure and arrangement, we need __

    What is the relationship between magnification and resolution?
    0.4 nm on a good light microscope

    oil immersion

    the higher the magnification, the less the resolution, the shorter the distance, and the less aperture there is
  4. What is aperture?
    The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance.
  5. All __ have a __, causing light.

    How many condenser lenses are there?
    condenser lenses 

    refractive index

    three types to bend at a minimum of 45-ish degrees
  6. When light is scattered, what happens?
    Less magnification means what?
    • less light goes up the tube
    • more light to collect
  7. How to combat the fact that light is scattered and less magnification occurs?

    Explain it in terms of a, b, and c.
    immersion oil lens

    A= has a certain RI

    B= shorter R.I. depending on viscosity

    C= 50:50 mixture of both

    Light goes through lens and through teh oil, therefore collecting more light
  8. Brightfield Illumination (Light)
    - Cells? 
    - Stains?
    - background light?
    - magnification?
    - opaque disk? dark?
    • - cells, for the most part, are clear
    • - We go from red to violet (ROYGBIV) for stains except for green
    • - All the background light enters the lens
    • - Looking at max 1000 times magnification
    • - With opaque disk, it removes/ dampens background light; only light reflected by specimen
    • - Dark: used to look at structures in relation to cell wall or plasma membrane
  9. Phase Contrast Microscopy
    based on principle that, if you keep the wavelength the same, but make them out of phase, to get finer detail/ structure

    --> in phase means moving identically (can't tell it apart)

    1400x: more magnification but not tremendous
  10. DIC Microscopy
    • out of phase
    • two light sources
    • possibly two prisms to split the light
  11. Fluorescence Microscopy (ungodly important)
    • tremendous enormous diagnostic tool
    • - we combine microbio, immuno, and physics
  12. With Fluorescent Microscopy, what is used?
    antibodies are used that can take up a fluorescent molecule; the arms are bound to the organism; reacts with rest of immune system
  13. Confocal Microscopy
    uses lasers to excite the fluorophores

    eight different fluorescent molecules work
  14. EM is the __ and __.

    What must occur with this?
    TEM and SEM

    the specimen must be thin with heavy salts
  15. Scanning EM
    - scans whole surface

    - electrons bounce off the specimen and activate the specimen--> surface topography seen
  16. Prep of Specimen for the Light Microscope
    Stains, such as methylene blue and Safranin 

    India ink: used to stain a slide containing micro-organisms. The background is stained while the organisms remain clear. This is called a negative stain. India ink, along with other stains, can be used to determine if a cell has a gelatinous capsule.[15]
  17. What is a mordant?
    used to hold a stain because stains themselves don't stick to the organism

    it is an ion that binds a chemical dye and holds it down such that the dye remains stuck on teh organism
  18. What is differential staining?
    staining processes which use more than one chemical stain. 

    Ex: gram stains
  19. Explain gram stains
    • Positive: multilayers thck that have alot of polypeptides
    • Negative: thin cell wall; no fancy polypeptides, weaving it through

    Gram stains exploint differences between strong and weak cell walls; two stains distinguish between positive and negative
  20. Iodine as a stain
    Iodine is also used as a mordant in Gram's staining, it enhances dye to enter through the pore present in the cell wall/membrane

    It is dark and does not really emit light so it doesn't manipulate the color

    It physically reacts with it, but doesn't change the color
  21. Third step in gram staining?
    • Decolorize: 
    • gram + and negative

    dehydrate but strip cell wall

    Gram positive should stay purple because of large cell wall. CV-I complex get in and can't get back out--> color remains

    Gram negative should not maintain purple because the CV-I complex gets washed out due to thin wall--> washed away-->counterstain

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