Transcript Micro Chapter 3 ppt 11th edition

Chapter 3
Observing
Microorganisms
Through a
Microscope
© 2013 Pearson Education, Inc.
Copyright © 2013 Pearson Education, Inc.
Lectures prepared by Christine L. Case
Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc.
Figure 3.2 Microscopes and Magnification.
Unaided eye
≥ 200 m
Light microscope
200 nm – 10 mm
Tick
Actual size
Scanning
electron
microscope
10 nm – 1 mm
Red blood cells
Transmission
electron
microscope
10 pm – 100  m
E. coli bacteria
T-even bacteriophages
(viruses)
Atomic force
microscope
0.1 nm – 10nm
DNA double helix
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Units of Measurement
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1 µm = 10–6 m = 10–3 mm
1 nm = 10–9 m = 10–6 mm
1000 nm = 1 µm
0.001 µm = 1 nm
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Microscopy: The Instruments
 A simple microscope has only one lens
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Figure 1.2b Anton van Leeuwenhoek’s microscopic observations.
Lens
Location of specimen
on pin
Specimen-positioning
screw
Focusing control
Stage-positioning screw
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Microscope replica
Light Microscopy
 The use of any kind of microscope that uses visible
light to observe specimens
 Types of light microscopy
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Compound light microscopy
Darkfield microscopy
Phase-contrast microscopy
Differential interference contrast microscopy
Fluorescence microscopy
Confocal microscopy
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Figure 3.1a The compound light microscope.
Ocular lens
(eyepiece)
Remagnifies the
image formed by
the objective lens
Fine focusing knob
Coarse focusing knob
Body tube
Transmits the
image from the
objective lens to
the ocular lens
Arm
Objective lenses
Primary lenses that
magnify the
specimen
Stage Holds the
microscope slide
in position
Condenser Focuses
light through specimen
Diaphragm Controls the
amount of light entering the
condenser
Illuminator Light source
Base
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Principal parts and
functions
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
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Figure 3.1b The compound light microscope.
Ocular lens
Line of vision
Path of light
Prism
Body tube
Objective
lenses
Specimen
Condenser
lenses
Illuminator
Base with
source of
illumination
The path of light
(bottom to top)
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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
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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
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Figure 3.3 Refraction in the compound microscope using an oil immersion objective lens.
Oil immersion
objective lens
Unrefracted
light
Without immersion oil
most light is refracted
and lost
Immersion oil
Air
Glass slide
Condenser
lenses
Condenser
Iris diaphragm
Light source
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Brightfield Illumination
 Dark objects are visible against a bright background
 Light reflected off the specimen does not enter the
objective lens
ANIMATION Light Microscopy
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Figure 3.4a Brightfield, darkfield, and phase-contrast microscopy.
Eye
Eye
Eye
Ocular lens
Ocular lens
Diffraction plates
Objective lens
Only light reflected
by the specimen is
captured by the
objective lens
Undiffracted light
(unaltered by specimen)
Objective lens
Unreflected light
Specimen
Refracted or diffracted
light (altered by
specimen)
Specimen
Condenser lens
Condenser lens
Opaque disk
Light
Brightfield
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Light
Annular diaphragm
Light
Darkfield Illumination
 Light objects are visible against a dark background
 Light reflected off the specimen enters the objective
lens
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Figure 3.4b Brightfield, darkfield, and phase-contrast microscopy.
Eye
Eye
Eye
Ocular lens
Ocular lens
Diffraction plates
Objective lens
Only light reflected
by the specimen is
captured by the
objective lens
Undiffracted light
(unaltered by specimen)
Objective lens
Unreflected light
Specimen
Refracted or diffracted
light (altered by
specimen)
Specimen
Condenser lens
Condenser lens
Opaque disk
Light
Light
Darkfield
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Annular diaphragm
Light
Phase-Contrast Microscopy
 Accentuates diffraction of the light that passes
through a specimen
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Figure 3.4c Brightfield, darkfield, and phase-contrast microscopy.
Eye
Eye
Eye
Ocular lens
Ocular lens
Diffraction plates
Objective lens
Only light reflected
by the specimen is
captured by the
objective lens
Undiffracted light
(unaltered by specimen)
Objective lens
Unreflected light
Specimen
Refracted or diffracted
light (altered by
specimen)
Specimen
Condenser lens
Condenser lens
Opaque disk
Light
Light
Annular diaphragm
Light
Phase-contrast
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Differential Interference Contrast
Microscopy
 Accentuates diffraction of the light that passes
through a specimen; uses two beams of light
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Figure 3.5 Differential interference contrast (DIC) microscopy.
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Fluorescence Microscopy
 Uses UV light
 Fluorescent substances absorb UV light and emit
visible light
 Cells may be stained with fluorescent dyes
(fluorochromes)
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Figure 3.6b The principle of immunofluorescence.
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Confocal Microscopy
 Cells are stained with fluorochrome dyes
 Short-wavelength (blue) light is used to excite the
dyes
 The light illuminates each plane in a specimen to
produce a three-dimensional image
 Up to 100 µm deep
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Figure 3.7 Confocal microscopy.
Nucleus
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Two-Photon Microscopy
 Cells are stained with fluorochrome dyes
 Two photons of long-wavelength (red) light are used
to excite the dyes
 Used to study cells attached to a surface
 Up to 1 mm deep
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Figure 3.8 Two-photon microscopy (TPM).
Food vacuoles
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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
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Figure 3.9 Scanning acoustic microscopy (SAM) of a bacterial biofilm on glass.
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Electron Microscopy
 Uses electrons instead of light
 The shorter wavelength of electrons gives greater
resolution
ANIMATION Electron Microscopy
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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
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Figure 3.10a Transmission and scanning electron microscopy.
Electron gun
Electron beam
Electromagnetic
condenser lens
Specimen
Electromagnetic
objective lens
Electromagnetic
projector lens
Fluorescent screen
or photographic
plate
Transmission
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Viewing
eyepiece
Primary electron beam
Electromagnetic lenses
Viewing
screen
Electron
collector
Secondary
electrons
Specimen
Amplifier
Transmission Electron Microscopy
(TEM)
 10,000–100,000; resolution 2.5 nm
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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
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Figure 3.10b Transmission and scanning electron microscopy.
Electron gun
Electron beam
Electromagnetic
condenser lens
Specimen
Electromagnetic
objective lens
Electromagnetic
projector lens
Fluorescent screen
or photographic
plate
Viewing
eyepiece
Electromagnetic lenses
Viewing
screen
Electron
collector
Secondary
electrons
Specimen
Amplifier
Scanning
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Primary electron beam
Scanning Electron Microscopy (SEM)
 1,000–10,000; resolution 20 nm
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Scanned-Probe Microscopy
 Scanning tunneling microscopy (STM) uses a
metal probe to scan a specimen
 Resolution 1/100 of an atom
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Figure 3.11a Scanned-probe microscopy.
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Scanned-Probe Microscopy
 Atomic force microscopy (AFM) uses a
metal-and-diamond probe inserted into the
specimen
 Produces three-dimensional images
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Figure 3.11b Scanned-probe microscopy.
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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
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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
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Preparing Smears for Staining
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Stains consist of a positive and negative ion
In a basic dye, the chromophore is a cation
In an acidic dye, the chromophore is an anion
Staining the background instead of the cell is called
negative staining
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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
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Differential Stains
 Used to distinguish between bacteria
 Gram stain
 Acid-fast stain
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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
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Gram Stain
Color of
Gram-Positive Cells
Color of
Gram-Negative Cells
Primary Stain:
Crystal Violet
Purple
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing Agent:
Alcohol-Acetone
Purple
Colorless
Counterstain:
Safranin
Purple
Red
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Figure 3.12b Gram staining.
Rod
(gram-negative)
Cocci
(gram-positive)
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Acid-Fast Stain
 Stained waxy cell wall is not decolorized by
acid-alcohol
 Mycobacterium
 Nocardia
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Acid-Fast Stain
Color of
Acid-Fast
Color of
Non–Acid-Fast
Primary Stain:
Carbolfuchsin
Red
Red
Decolorizing Agent:
Acid-alcohol
Red
Colorless
Counterstain:
Methylene Blue
Red
Blue
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Figure 3.13 Acid-fast bacteria.
M. bovis
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Special Stains
 Used to distinguish parts of cells
 Capsule stain
 Endospore stain
 Flagella stain
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Negative Staining for Capsules
 Cells stained
 Negative stain
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Figure 3.14a Special staining.
Capsules
Negative staining
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Endospore Staining
 Primary stain: malachite green, usually with heat
 Decolorize cells: water
 Counterstain: safranin
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Figure 3.14b Special staining.
Endospore
Endospore staining
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Flagella Staining
 Mordant on flagella
 Carbolfuchsin simple stain
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Figure 3.14c Special staining.
Flagellum
Flagella staining
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