Block One Text 4

  1. a.       When measuring microbes, we use the __ and the __ as the __
    b.      Microorganisms are measured in __(equals 0.000001 m) or __( equals 0.000000001 [10-9])
    metric system

    meter (m) as the standard unit


  2. a.       __used a microscope with only one lens, but his single lens could magnify __
    b.      __ s credited with making the first compound microscope around 1600; they were poor quality and could not see bacteria
    c.       __ developed a better one in 1830; and, with improvements, the modern CM was formed
    • Leeuwenhoek 
    • 300 x

    • Zaccharias Janssen
    • Joseph Jackson Lister (father of J. Lister)
  3. What is light microscopy?

    Explain a modern compound light microscope.

    What is a CLM used for?
    refers to the use of any kind of microscope that uses visible light to observe specimens

    has a series of lenses and uses visible light as its source of illumination

    to examine small specimen and their fine detail
  4. Parts of CM
                                                                   i.      A series of finely ground lenses forms a clearly focused image that is achieve when light rays from an illuminator, passes though a condenser, which has lenses that direct the light rays through the specimen. From here, light rays pass into the objective lenses, the lenses closest to the specimen. The image of the specimen is magnified again by the ocular lens, or eyepiece
  5. e. Calculating total magnification

    Values of objective lens? Ocular lens? 

    Total magnification in relation of the lens
    • a.       multiplying the objective lens magnification (power) by the ocular lens magnification (power)
    •                                                                i.      Objective lenses can be 10 X (low power), 40 X (high power), and 100 X (oil immersion)
    •                                                              ii.      Ocular lenses magnify specimens by a factor of 10
    •                                                             iii.      Total magnification would be 100X for low, 400X for high, and 1000X for oil immersion
  6. Resolution

    Relation to wavelength
    • a.       is the ability of the lenses to distinguish fine detail and structure, or the ability of the lenses to distinguish two points a specified distance apart
    •                                                                i.      The shorter the wavelength of light used in the instrument, the greater the resolution
  7. a.       To obtain a cear, finely detailed image under a compound light microscope, specimens must be made to __
                                                                   i.      To obtain such contrast, we must change the __ of specimens from that of their medium
    • contrast sharply with their medium
    • refractive index
  8. What is RI?
    •                                                                i.      a measure of the light-bending ability of a medium
    • 1.       We change the refractive index of specimens by staining
  9. a. Light rays move in a straight line through a single medium. After the specimen is stained, when light ras pass through the two materials with different refractive indexes, what happens to the rays?  As the light rays travel away from the specimen, they spread out and enter the objective lens, and the image is magnified
    the rays change direction (refract) from a straight path by bending or changing angle at the boundary between the materials and increase the image’s contrast between the specimen and the medium.
  10. a.       To achieve high magnification (1000X) with good resolution, the __ must be small.

    What can be used? Why?
    objective lens

    immersion oil 

    it has the same RI as glass, so the oil becomes part of the optics of the glass of the microscope
  11.                                                                                                                                        i.      Unless immersion oil is used, light rays are __as tey enter the air from the slide, and the objective lens would have to be __ to capture most of them
                                                                                                                                         ii.      The oil has the same effect as __; therefore, it improves the resolving power of the lenses
    b.      If oil is not used with an oil immersion objective lens, the image becomes __
    • refracted 
    • increased in diameter

    • increasing the objective lens diameter
    • fuzzy, with poor resolution
  12.                                                                i.      Under usual operating conditions, the field of vision in a compound LM is brightly illuminated. By focusing the light, the condenser produces a __
    brightfield illumination
  13. Darkfield Microscope: 

    - use? 

    - components?

    - how it works? 

    - how the specimen looks?
    •                                                                i.      Use: to examine live microorganisms that either are invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics then cannot be identified; used to examine unstained microorganisms suspended in liquid
    • 1.       Example: examining very thin spirochetes
    •                                                              ii.      Components: Instead of a normal condenser, it uses a darkfield condenser that contains an opaque disk, which bocks light that would enter the objective lens directly
    •                                                             iii.      How it works: only light that is reflected off (turned away from) the specimen enters the ojective lens
    •                                                            iv.      How the specimen looks: because there is no direct background light, the specimen appears light against a black background—the dark field
  14. Phase Contrast Microscopy
    •                                                                i.      Use: permits detailed examination of internal structures in living microorganisms; not necessary to fix or stain the specimen, which could both distort/ kill the specimen
    •                                                              ii.      How it works: based on wave nature of light rays and the fact that light rays can be in phase or out of phase
  15. Explain in detail how Phase contrast microscopy works.
    • Reinforcement: waves are coinciding with each other
    • Interference (relative darkness): Waves are out of sync

    One set of light rays comes from light source; the other is reflected from structure in specimen. 

    When two sets of light rays are brought together, the form an image on the oculr lens, contianing areas of light (in phase) and grayish areas (out of phase)
  16. How the specimen looks in phase contrast microscopy.
    the internal structres of the cell become more sharply defined
  17. l. Differential Interference Contrast (DIC) Microscopy

    i. How it works
    ii. How the specimen looks
    •                                                                i.      How it works: similar to phase-contrast microscopy in that it uses differences in RIs except it uses two beams of light instead of one. Also, prisms split each light beam, adding contrasting colors to the specimen; resolution is higher than that of a standard phase-contrast microscope
    •                                                              ii.      How the specimen looks: image is brightly colored and appears nearly 3D
  18. a.       Fluorescence Microscopy
                                                                   i.      Use: __
    1.       Some organisms fluoresce naturally under UV light; if not, it is stained with __
    2.       The principle use is a diagnostic technique called the __
    uses fluorescence, the ability of substances to absorb short wavelengths of light (UV) and give off light at a longer wavelength (visible)

    fluorochromes (fluorescent dyes)

    fluorescent-antibody technique, or immunofluorescence
  19. a.       Fluorescent antibodies for a particular antigen are obtained as follows:
    i.      An animal is injected with a specific antigen, and the animal then begins to produce antibodies against that antigen. After a sufficient time, the antibodies are removed from the serum of the animal. Next, a fluorochrome is chemically combined with the antibodies, which are then added to a microscopic slide containing an unknown bacterium
  20. 1.       This technique can __ or __, even within cells, tissues, or other pathogenic microorganisms
    2.       Most importantly, it can be used to identify a microbe in minutes
    detect bacteria or other pathogenic microorganisms
  21.                                                                i.      How it works: __
                                                                 ii.      How the specimen looks: __
    fluorochromes have special attractions for different microorganisms

    they appear as luminescent, bright objects against a dark background
  22. a.       Confocal Microscopy
                                                                   i.      Uses: __
    1.       The scanned planes of a specimen are converted to a digital form that can be used by a computer to construct a 3D representation and can be rotated and viewed
    2.       Can be used to evaluate __ by monitoring the distributions and concentrations of substances like ATP and calcium ions
                                                                 ii.      Components: __
    used to reconstruct 3D images

    cellular physiology

    uses a pinhole aperture to eliminate blurring
  23. Confocal Microscope:
          How it works: __
    1.       Instead of illuminating the entire field, ___
    2.       Each plane corresponds to an image of a fine slice that has been physically cut from a specimen. Successive planes and regions are illuminated until the entire specimen has been scanned
    specimens are stained with fluorochromes so they will emit, or return, light

    one plane of a small region of a specimen is illuminated with a short wavelength light which passes the returned light through an aperture aligned with the illuminated region
  24. a.       Electron Microscopy
                                                                   i.      Uses: __
                                                                 ii.      How it works: __
                                                                iii.      Components: __ to focus a beam of electrons
    used to see smaller objects that cannot be seen with LMs

    a beam of electrons is used, which travel in waves; the resolving power is greater than other microscopes and due to shorter wavelengths of electrons

    electromagnetic lenses
  25. Types
    • SEM
    • TEM:  a finely focused beam of electrons from an electron gun passes through a specially prepared, ultrathin section of the specimen; the beam is focused by the electromagnetic condenser lens that is similar to condenser of LM
  26. TEM

    a.       Uses: __

    Disadvantages: __
    Has high resolution and is valuable for examining different layers of specimens

    only a thin section can be usedà no 3D aspects; specimens must be fixed, dehydrated, and viewed under a high vacuumà kills and shrinks/ distorts specimen and results in artifacts
  27. a.       Components: uses __ to control illumination, focus, and magnification

    b.      How it works: specimen placed on a __; beam of electrons pass through specimen--> __--> focused by an __ onto a fluorescent screen or photographic plateà final image __
    electromagnetic lenses

    copper mesh grid

    electromagnetic objective lens

    electromagnetic projector lens

    (transmission electron micrograph)
  28. EM:
    Can resolve objects as close together as __
    ii.    Contrast can be resolved by __ that __ and produce __.
    1.     Metals can be fixed onto the specimen (__) or used to increase the electron opacity of the surrounding field (__), which is used for virus particles, bacterial flagella, and protein molecules
    • 10 pm
    • stains that absorb electrons and produce a darker image
    • positive staining
    • negative staining
  29. How the specimen looks on an EM
    • a.       can be viewed with shadow casting:
    •                                                                                                                                       i.      Heavy metal is sprayed at an angle so it strikes the microbe from one side. The uncoated area leaves a clear area behind it as a shadow, giving a 3D effect
  30. SEM:
    How it work: __
                                                                                                                                          i.      An electron gun produces a finely focused beam of electrons called the __, which pass through __ and are directed over the surface of the specimen
    1.       The primary electron beam __, and the secondary electrons produced are __, amplified, and used to produce an image on a viewing screenà __
    • provides 3D views of specimens
    • primary electron beam
    • electromagnetic lenses
    • knocks electrons out of the surface of the specimen
    • transmitted to an electron collector
    • scanining electron micrograph
  31. SEM: a.       Uses: __
    useful in studying the surface structures of intact cells and viruses
  32. a.       Smears for Staining (what is it?)
                                                                  i.      Process:
    1.       __
    (coloring the specimen with a dye to emphasize structures)

    • fix to slide (which kills it but preserves natural state)
    • a.       When set to be fixed, a thin film of material with the microorganisms is spread over the surface of the slide, which is called a smear, and allowed to air dry or run through Bunsen burner
    • 2.       Wash off stain and blot
  33.                                                               i.      Stains are salts with __ and __ ion, one of which is colored and called a __
    1.       The color of basic dyes is in the __; in acidic dyes, it is the __
    a.       Because bacteria are __, the colored __ is attracted to the cell (crystal violent, blue, green, etc.)
    • positive and negative ions
    • chromophore
    • positive ion
    • negative ion
    • negative
    • positive ion in a basic dye
  34.                                                               i.      Preparing colorless bacteria against a colored background is called __ and is useful for making the specimen visible against a dark background with minimal distortion of cell and size and shape
    negative staining
  35.                                                               i.      To apply acidic or basic des, there are three staining techniques:

    • 1) Simple
    • 2) Differential
    • 3) Acid fast
  36. Explain simple stain
    • an aqueous or alcohol solution of a single basic dye
    • a.       Different dyes bind specifically to different parts of cells; the primary purpose of a simple stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible
  37. 1.       Chemicals can be added to the solution to intensify the stain, c a _alled_; What does it do?
    • mordant
    • it increases the affinity of a stain for a biological specimen; another is to cot a structure to make it thicker and easier to see after it is stained with a dye
  38. 1.       Differential Stains:
    react differently with different kinds of bacteria and used for distinguishing
  39. Procedure of gram stain
                                                                                                                                          i.      Procedure: heat fixed smear covered with basic purple dye (primary stain)à washed offà smear covered with iodineà both look dark violet or purpleà washed with alcohol or an alcohol-acetone solution (decolorizing agent)à alcohol rinsed off and slide stained with safranin (basic red dye)à smear washed again, blotted, and examined
  40. Classifying gram stains
                                                                                                                                          i.      Classifying: after the purple dye and iodine (which color the cytoplasm of each bacterium dark violet or purple) has been decolorized, any bacteria that retain this color are gram-positive. Those that do not are gram-negative
  41. Why is safranin used?
    a.       Stains such as safranin that have a contrasting color to the primary stain are called counterstains. Because gram-positive bacteria retain the original purple stain, the are unaffected by the safranin counterstain
  42. What are the differences due to?
    thicker peptidoglycan wall in gram positive bacteria

    gram negative bacteria contain a layer of lipopolysaccharide as part of their cell wall

    •                                                                                                                                       i.      When applied to both gram-positive and gram-negative cells, crystal violet and then iodine readily enter the cells, which, once inside, combine to form VI-I, which cannot be washed out of the intact peptidoglycan laer of gram-positive cels by alcohol due to size
    • 1.       Consequently, gram-positive cells retain the color of the crysal violet dye; gram-negative cells have it washed out by alcohol, which disrupts the outer lipopolysaccharide layer
    • 2.       Summary: gram-positive retain de and appear purple; gram-negative do not and are colorless until counterstained with red 
  43. a.       Gram-positive bacteria can be killed easily by __ and __
    b.      Gram-negative bacteria are __ because the antibiotics cannot penetrate the __
    • penicillins and cephalosporins
    • more resistant
    • lipopolysaccharide layer

  44. a.       Acid-Fast Stain: b__
    binds strongly only to bacteria that have a waxy material in their cell walls and used to identify Mycobacterium types, as well as the pathogenic stains of the genus Nocardia
  45. Special Stains: __
    used to color and isolate specific parts of microorganisms, like endospores, and to reveal capsules
  46.                                                                                                                                       i.      Negative Staining for Capsules
    1.       Many microorganisms contain a __; and, demonstrating the presence of a __is a means of determining the organism’s virulence, the degree to which a pathogen can cause disease

    1.       Capsule staining= difficult due to __--> may be removed during washing
    • gelatinous covering called a capsule
    • capsule

    materials being soluble in water
  47. What is an endospore?
    1.       An endospore is a special resistant, dormant  structure formed within a cell that protects a bacterium from adverse environmental conditions
  48.                                                                                                                                       i.      Flagella Staining
    1.       : a mordant is used and the stain carbolfuchsin to build up diameters of flagella until visible
Card Set
Block One Text 4
Test One