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Chapter 2
Lecture Outline
Observing the
Microbial Cell
Observing Microbes

Visible light has wavelengths of 400–750 nm
 Maximum

Human eyes have limited resolution
 150

resolution is 1 wavelength
mm (1/7 mm, 1/200 inch)
Magnification spreads light rays out
 To

150 mm, resolution
of our eyes
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Distance between
2
Observing Microbes

Microscope needed to see smaller objects
 Eukaryotic
microbes
Protozoa, algae, fungi
 10–100 mm

 Prokaryotes
Bacteria, Archaea
 0.4–10 mm

10 mm
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Relative Sizes of Different Cells
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Types of Microscopes

Light microscopy
 Resolves
images of individual bacteria based on their
absorption of light


Bright field, dark field, phase contrast, fluorescence
Electron microscopy
 Uses
beams of electrons to resolve details several
orders of magnitude smaller than those seen under
light microscopy


Scanning, transmission
Atomic force microscopy
 Uses
van der Waals forces between a probe and
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object to map the
topography of a cell
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Microscopy: Range of Resolution
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Bacterial Shapes

Cocci = spheres
 in

bunches
quartets
Bacilli = sticks, rods
 alone

chains
in chains
Vibrios = bent rods


Spiral
Irregular
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Bright-Field Microscopy

Increasing resolution
 Use

shorter wavelength light
UV, X-rays

But images aren’t visible to human eye
 Lessen

contrast
Lenses with higher contrast give less resolution

But need enough contrast to see object
 Immersion

Collects more light from specimen
 Wider

oil
lens closer to specimen
Higher numerical aperture (NA)
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NA = n sin
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Bright-Field Microscopy

Increasing resolution
 Multiple
lenses
Correct each other’s aberrations
 Compound microscope
 Need to focus two lenses

Objective
 Condenser

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Staining
Fix cells to hold in position
 Stain with dye

 Reacts

with chemical structure of organism
Gram stain reacts with thick cell wall
 Increases

absorbance
Easier to find in low-contrast conditions
Gram-negative cells
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Gram-positive cells
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Dark-Field Microscopy

Light shines at oblique angle
 Only
light scattered by sample
reaches objective
 With enough light, some
bounces off object

Even objects smaller than wavelength of light
 Makes

visible objects below resolution limit
Flagella, very thin bacteria
Helical bundle of flagella
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Phase-Contrast Microscopy
Light passes through and
around sample
 Light through sample is refracted

 Changes
phase of light
 Light waves out of phase cancel

Sample appears dark against
light background
 Shows
internal
organelles of eukaryotes
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Differential Interference
Contrast (DIC) Microscopy

Polarized light passes through specimen
 Sample
boundaries bend light
 Second polarized lens blocks light
 Bent light affects brighter or darker than
Cell nuclei
background
Head of microscopic
worm (C. elegans)
Bacterium
Pharynx (mouth)
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10 mm
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Fluorescence Microscopy

Fluorophores absorb high-energy light
 Short

wavelength
Emit lower-energy light
 Longer

wavelength
UV
blue
green
blue
green
red
Label molecules of interest in cell
 Marker
for position of molecules within cell
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Fluorescently Labeling Molecules

Attach directly to some molecules
 DAPI

binds DNA
Attach labeled antibody to molecules
 Antibody

binds specific molecules
Fluor covalently bound to antibody
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Fluorescently Labeling Molecules

Attach labeled nucleotides to DNA
 Nucleotide


probe base-pairs to DNA
Fluor covalently bound to probe
Gene fusion
 Protein

of interest fused to fluorescent protein
GFP from jellyfish
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Electron Microscopy

Electrons behave like light waves
 Very
high frequency
 Allows very great resolution


A few nanometers
Sample must absorb electrons
 Coated
with heavy metal
 Electron beam and sample are
in a vacuum
 Lenses are magnetic fields
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Transmission EM

Sample is fixed to prevent protein movement
 Aldehydes
to fix proteins
 Flash-freezing
 High-intensity microwaves

Fixed sample is sliced very thin
 Microtome

Sample is stained with metal
 Uranium
 Osmium
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Transmission EM

High resolution
 Can
detect molecular
complexes
Ribosomes
 Flagellar base
 Strands of DNA


Need many slices to
determine 3D structure
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Scanning EM

Sample is coated with
heavy metal
 Not
sliced
 Retains 3D structure
 Gives 3D image

Only examines
surface of sample
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Atomic Force Microscopy
Concept Quiz
The best technique for examining the
presence of a structure with a diameter of
500 nm is
light microscopy.
b. electron microscopy.
c. DIC microscopy.
a.
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Concept Quiz
A light microscopic technique that can
visualize structures below the resolution limit
of light is
dark-field microscopy.
b. phase-contrast microscopy.
c. differential interference contrast
microscopy.
a.
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Concept Quiz
The best technique for examining the
presence of a chemical structure with a
diameter of 3 nm is
light microscopy.
b. electron microscopy.
c. DIC microscopy.
a.
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