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Ch3-Observing Organisms Through A Microscope

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CH3-OBSERVING ORGANISMS THROUGH A MICROSCOPE * * * * * * * * * * * * * Red * * * * * Q&A Acid-fast staining of a patient s sputum is a rapid, reliable, and ... – PowerPoint PPT presentation

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Title: Ch3-Observing Organisms Through A Microscope


1
Ch3-Observing Organisms Through A Microscope
2
Units of Measurement
  • 1 µm 106 m 103 mm
  • 1 nm 109 m 106 mm
  • 1000 nm 1 µm
  • 0.001 µm 1 nm

3
Microscopy The Instruments
  • A simple microscope has only one lens

4
Light Microscopy
  • Use of any kind of microscope that uses visible
    light to observe specimens
  • Types of light microscopy
  • Compound light microscopy
  • Darkfield microscopy
  • Phase-contrast microscopy
  • Differential interference contrast microscopy
  • Fluorescence microscopy
  • Confocal microscopy

5
The Compound Light Microscope
6
Compound Light Microscopy
  • In a compound microscope, the image from the
    objective lens is magnified again by the ocular
    lens
  • Total magnification objective lens ? ocular
    lens
  • Parfocal microscope objectives stay in focus when
    magnification is changed i.e., if the microscope
    is switched from a higher power objective (e.g.,
    40) to a lower power objective (e.g., 10), the
    object stays in focus.

7
Compound Light Microscopy
  • Resolution is the ability of the lenses to
    distinguish two points
  • A microscope with a resolving power of 0.4 nm can
    distinguish between two points 0.4 nm
  • Shorter wavelengths of light provide greater
    resolution
  • Resolving powerWavelength of illumination/2 X
    numerical aperture

8
Compound Light Microscopy
  • The refractive index is a measure of the
    light-bending ability of a medium
  • The light may bend in air so much that it misses
    the small high-magnification lens
  • Immersion oil is used to keep light from bending
  • Numerical aperture for oil immersion objective
    1.3
  • In optics, chromatic aberration (also called
    achromatism) is the failure of a lens to focus
    all colors to the same point. It occurs because
    lenses have a different refractive index for
    different wavelengths of light (the dispersion of
    the lens).

9
Refraction in the Compound Microscope
chromatic aberration can be overcome by the use
of a compound microscope, in which the image is
relayed by two lens arrays.
10
Darkfield Illumination
  • Light objects are visible against a dark
    background
  • Light reflected off the specimen enters the
    objective lens
  • Used to view specimens that cant be stained by
    standard methods (ex Treponema pallidum)

11
Phase-Contrast Microscopy
  • Accentuates diffraction of the light that passes
    through a specimen
  • Used to view the internal structure of living
    microorganisms

12
Differential Interference Contrast Microscopy
  • Accentuates diffraction of the light that passes
    through a specimen uses two beams of light

13
Fluorescence Microscopy
  • Uses UV light
  • Fluorescent substances absorb UV light and emit
    visible light
  • Cells may be stained with fluorescent dyes
    (fluorochromes)
  • Used to identify pathogenic bacteria in clinical
    specimens

14
Confocal Microscopy
  • Cells stained with fluorochrome dyes
  • Short wavelength (blue) light used to excite the
    dyes
  • The light illuminates each plane in a specimen to
    produce a three-dimensional image
  • Up to 100 µm deep

15
Two-Photon Microscopy
  • Cells stained with fluorochrome dyes
  • Two photons of long- wavelength (red) light used
    to excite the dyes
  • Used to study cells attached to a surface
  • Up to 1 mm deep

16
Scanning Acoustic Microscopy (SAM)
  • Measures sound waves that are reflected back from
    an object
  • Used to study cells attached to a surface
  • Resolution 1 µm

17
Electron Microscopy
  • Uses electrons instead of light
  • The shorter wavelength of electrons gives greater
    resolution
  • Used to view objects smaller than 0.2 µm (ex
    viruses)

ANIMATION Electron Microscopy
18
Transmission Electron Microscopy (TEM)
  • Ultrathin sections of specimens
  • Light passes through specimen, then an
    electromagnetic lens, to a screen or film
  • Specimens may be stained with heavy metal salts

19
Transmission Electron Microscopy (TEM)
  • 10,000100,000? resolution 2.5 nm

20
Scanning Electron Microscopy (SEM)
  • An electron gun produces a beam of electrons that
    scans the surface of a whole specimen
  • Secondary electrons emitted from the specimen
    produce the image

21
Scanning Electron Microscopy (SEM)
  • 1,00010,000? resolution 20 nm

22
Scanned-Probe Microscopy
  • Scanning tunneling microscopy (STM) uses a metal
    probe to scan a specimen
  • Resolution 1/100 of an atom

23
Scanned-Probe Microscopy
  • Atomic force microscopy (AFM) uses a metal-
    and-diamond probe inserted into the specimen.
  • Produces three-dimensional images.

24
Preparing Smears for Staining
  • Staining Coloring the microbe with a dye that
    emphasizes certain structures
  • Smear A thin film of a solution of microbes on a
    slide
  • A smear is usually fixed to attach the microbes
    to the slide and to kill the microbes

25
Preparing Smears for Staining
  • Live or unstained cells have little contrast with
    the surrounding medium. Researchers do make
    discoveries about cell behavior by observing live
    specimens.

ANIMATION Microscopy and Staining Overview
26
Preparing Smears for Staining
  • Stains consist of a positive and negative ion
  • In a basic dye, the chromophore is a cation
  • bacteria are negatively charged at pH 7 so the
    basic dye is attracted to the bacteria
  • In an acidic dye, the chromophore is an anion
  • Staining the background instead of the cell is
    called negative staining

27
Simple Stains
  • Simple stain Use of a single basic dye
  • A mordant may be used to hold the stain or coat
    the specimen to enlarge it

ANIMATION Staining
28
Differential Stains
  • Used to distinguish between bacteria
  • Gram stain
  • Acid-fast stain

Gram-stained Bacteria
29
Gram Stain
  • Classifies bacteria into gram-positive or
    gram-negative
  • Gram-positive bacteria tend to be killed by
    penicillin and detergents
  • Gram-negative bacteria are more resistant to
    antibiotics

30
Gram Stain
31
Acid-Fast Stain
  • Stained waxy cell wall is not decolorized by
    acid-alcohol (gram staining does not work with
    these 2 types of bacteria)
  • Mycobacterium
  • Nocardia

32
Acid-Fast Stain
33
Acid-Fast Bacteria
34
QA
  • Acid-fast staining of a patients sputum is a
    rapid, reliable, and inexpensive method to
    diagnose tuberculosis. What color would bacterial
    cells appear if the patient has tuberculosis?

35
Special Stains
  • Used to distinguish parts of cells
  • Capsule stain capsules do not accept most dyes,
    so Nigrosin dye or India ink must be used
  • Capsules surround certain bacteria make them
    more resistant to our immune systems (thus they
    are more virulentcause disease)
  • Endospore stain endospores protect bacteria in
    adverse environments resist normal dyes, so
    Schaeffer-Fulton endospore stain is used
  • Flagella stain too small to be seen w/out
    stains, so carbolfuchsin builds up the layers to
    be seen

36
Negative Staining for Capsules
  • Cells stained
  • Negative stain

37
Endospore Staining
  • Primary stain Malachite green, usually with heat
  • Decolorize cells Water
  • Counterstain Safranin

38
Flagella Staining
  • Mordant on flagella
  • Carbolfuchsin simple stain
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