Chapter 2
Observing the microbial cell
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Chapter Overview
How microorganisms are observed
● The bright-field microscope
● Staining bacterial cells
● The dark-field and phase-contrast
● The fluorescence and electron
microscopes
●
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Introduction
Since Leeuwenhoek’s time, powerful
microscopes have been devised to search
for microbes in unexpected habitats.
- Example = The human stomach
- Microscopy revealed
the presence of
Helicobacter pylori,
the cause of stomach
ulcers.
Figure 2.1
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Observing Microbes
The size at which objects become visible
depends on the resolution of the
observer’s eye.
Resolution is the smallest distance
between two closely placed objects that
can still be distinguished.
The resolution of the human retina is about
150 mm (1/7 mm).
Figure 1.1
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Microscopy: The Instruments
• 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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Detection is the ability to determine the
presence of an object (can not resolve individual
objects)
Magnification means an increase in the
apparent size of an image to resolve
smaller separations between objects.
Figure 2.3
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Microbial Size
Microbes differ in size, over a range of a few
orders of magnitude, or powers of ten.
- Eukaryotic microbes
- Protozoa, algae, fungi
- 10–100 mm
- Prokaryotes
- Bacteria, Archaea
- 0.4–10 mm
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Figure 2.4
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Microbial Shapes
Certain shapes of bacteria
are common to many
taxonomic groups.
- Bacilli = Rods
- Cocci = Spheres
- Spiral forms
- Spirochetes
- Spirilla
Figure 2.6
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Unusual Shapes of microorganisms
Stella
Haloarcula
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Microscopy for Different Size Scales
Different microscopes are required to resolve
various cells and subcellular structures.
Figure 2.7
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Optics and Properties of Light
Light is part of the spectrum of electromagnetic
radiation.
- Wavelength of visible light = 400–750 nm
For electromagnetic radiation to resolve an object,
certain conditions must exist:
1. Contrast between object and its medium
2. Wavelength smaller than the object
3. A detector with sufficient resolution for the
given wavelength
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Figure 2.8
13
Light Interacts with an Object
Absorption means that the photon’s energy is
acquired by the absorbing object.
Reflection means that the wave front bounces off
the surface of an object.
Refraction is the bending of light as it enters a
substance that slows its speed.
Scattering occurs when the wave front interacts
with an object smaller than the wavelength of
light.
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Figure 2.9
15
Wave fronts of light shift
direction as they enter
a substance of higher
refractive index.
Parabolic lenses bring
light rays to a focal
point.
Figure 2.10
Figure 2.11
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Generating an Image with a Lens
Figure 2.12
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Microscopes
•
•
•
•
•
•
Compound light microscope  White light
Fluorescence microscope  UV light
Confocal microscope  Laser light
Electron microscopeBeam of electrons
Magnification
Resolution
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The Compound Microscope
A system of multiple lenses designed to correct or
compensate for aberration
- Ocular lens
- Objective lens
- Needs to be parfocal
Total magnification = Magnification of ocular
multiplied by that of the objective
Empty magnification = Magnification without an
increase in resolution
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Compound Microscope
• 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
Figure 2.17
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Compound Light Microscope
 Applications:
Bright
field microscopy
Dark field microscopy
Phase-contrast microscopy
Fluorescence microscopy
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Bright-Field Microscopy
Generates a dark image of an object over a light
background
To increase resolution:
- Use shorter wavelength light
- Improve contrast
- Use immersion oil
- Use wider lens closer to specimen
- Higher numerical aperture (NA)
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Figure 2.15
Figure 2.16
NA = n sin
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Fixation and Staining
The detection and resolution of cells under
a microscope are enhanced by:
- Fixation = Cells are made to adhere to
a slide in a fixed position
- Staining = Cells are given a distinct
color
- Most stains have conjugated double
bonds or aromatic rings, and one or
more positive charges.
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A simple stain adds
dark color
specifically to cells,
but not to the
external medium or
surrounding tissue.
Figure 2.20
- Most commonly
used stain is
methylene blue.
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A differential stain stains one kind of cell
but not the other.
- Gram stain differentiates between two
types of bacteria.
- Gram-positive retain the crystal violet
stain because of their thicker cell wall.
- Gram-negative bacteria do not.
Figure 2.21
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Figure 2.22
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Basic Dyes used in Bacterial Staining
Crystal Violet
Safranin
Eosin Y
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Other differential stains
- Acid-fast stain = Carbolfuchsin used to stain
Mycobacterium species (M. tuberculosis and M.
leprae
- Spore stain = Malachite green used to detect
spores of Bacillus and Clostridium
- Negative stain = Colors the background, which
makes capsules more visible
Figure 2.24
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Special Stains
• Negative staining is
useful for capsules.
• Heat is required to
drive a stain into
endospores.
• Flagella staining
requires a mordant to
make the flagella
wide enough to see.
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Figure 3.12a-c
Dark-Field Microscopy
Dark-field optics enables microbes to be
visualized as halos of bright light against
darkness.
Light shines at oblique angle.
- Only light scattered by sample reaches
objective.
- Makes visible objects below resolution
limit
- Flagella, very thin bacteria
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Figure 2.25
Figure 2.27
Figure 2.26
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Phase-Contrast Microscopy
Superimposes refracted light and transmitted
light shifted out of phase
- Reveals differences in refractive index as
patterns of light and dark
- Can be used to view live cells and cellular
organelles
Figure 2.28
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Fluorescence Microscopy
In fluorescence microscopy, incident light is
absorbed by the specimen and reemitted at
a lower energy, thus longer wavelength.
Figure 2.31
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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
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Confocal Microscopy
In confocal laser scanning microscopy, both
excitation light and emitted light are focused
together.
-Can visualize cells in three dimensions
-Allows observation of live microbes in real time
Figure 2.34
Figure 2.35
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Confocal Microscopy
• Uses fluorochromes
and a laser light.
• The laser illuminates
each plane in a
specimen to produce
a 3-D image.
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Figure 3.7
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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Electron Microscopy
Two major types
- Transmission electron microscopy (TEM)
- Electrons pass through the specimen.
- Reveals internal structures
- Scanning electron microscopy (SEM)
- Electrons scan the specimen surface.
- Reveals external features in 3-D
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42
Figure 2.38
The TEM closely
parallels the
design of the
bright-field
microscope.
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Figure 2.39
The SEM is
arranged
somewhat
differently
from the TEM.
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Sample Preparation
The specimens for electron microscopy can be prepared in
several ways.
- Embedded in a polymer for thin sections
- Microtome is used to cut slices.
- Sprayed onto a copper grid
The specimen is then treated with a heavy-metal salt such
as uranyl acetate.
Note: For SEM, specimen is coated with heavy metal and it
is not sliced.
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Figure 2.40
Figure 2.41
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Cryo-Electron Microscopy
In cryo-EM, or electron cryo-microscopy,
the specimen is flash-frozen.
- Suspended in water and frozen rapidly in
a refrigerant
Cryo-electron tomography, or electron
cryotomography, avoids the need to
physically slice the sample for thin-section
TEM.
- Generates high-resolution models of
virus particles
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Chapter Summary
●
When observing microbes, resolution and
magnification are paramount.
●
Different kinds of microscopes are required to
resolve cells and subcellular structures:
- Bright-field: Employs various stains
- Dark-field: Detects unresolved objects
- Phase-contrast: Exploits differences in refractive
indices
- Fluorescence: Employs fluorophores for labeling
- Confocal: Visualizes cells in 3-D
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Chapter Summary
●
Electron microscopes use beam of electrons
instead of light rays.
- TEM: Provides internal details in 2-D
- SEM: Provides external details in 3-D
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