Chapter 1: Microscopy

• Discovering Cell Structure: Light Microscopy

• Improving Contrast in Light Microscopy
• Imaging Cells in Three Dimensions
• Probing Cell Structure: Electron Microscopy
Relative Sizes of Objects

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Some Principles of Light Microscopy

• Compound light microscope uses visible light to

illuminate cells
• Many different types of light microscopy:
• Bright-field
• Phase-contrast
• Dark-field
• Fluorescence
 Some Principles of Light Microscopy
• Bright-field scope (Figure 2.1a)
• Specimens are visualized because of
differences in contrast between Ocular Specimen
lenses on
specimen and surroundings glass slide

• Two sets of lenses form the image

Objective lens
(Figure 2.1b)
Stage

• Objective lens (usually 10x -100x mag.) & Condenser

ocular lens (usually 10x – 20x mag.) Focusing knobs
Light source
• Total magnification = objective
magnification ✕ ocular magnification
• Maximum magnification is ~2,000✕ (Figure 1.15 a)
Magnification Light path
100X, 400X, Visualized
1000X image
Eye

10X Ocular lens

Intermediate
image (inverted
from that of the
specimen)

10X, 40X or Objective lens

100X(oil)
Specimen

None Condenser lens

Light source
Figure 1.15 b
Some Principles of Light Microscopy

• Magnification: the ability to make an object larger

• Resolution: the ability to distinguish two adjacent
objects as separate and distinct
• Limit of resolution for light microscope is about
0.2 μm

What this means objects that are closer together

than 0.2 μm cannot be resolved
Calculating magnification
• Magnification = ocular x objective
• ex. Ocular = 10x, objective = 40x
• Magnification = 10 × 40 = 400x

Resolution
• The ability of a lens to distinguish
small objects that are close together
ex. resolving power of 0.2mm
• Two points can be distinguished if
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• Light must pass between two points for them to be viewed as

separate objects
• As wavelength decreases resolution improves.
Effect of Wavelength on Resolution
Throw ink-covered
objects at target (“E”):
Cannot fit between
arms, poor resolution

Fit between arms,

resolution improves

As diameter of objects
thrown decreases,
greater numbers pass
between the arms & the
resolution increases

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Improving Contrast in Light Microscopy

• Improving contrast results in a

better final image
• Staining improves contrast
• Dyes are organic compounds
that bind to specific cellular
materials
• Examples of common stains are
methylene blue, safranin, and
crystal violet
Figure 1.16
Simple staining – One dye used to color
specimen
• Chromophore – colored portion of a dye

Two types:
• Basic dye – positively charged
chromophore Crystal violet –
• Binds to negatively charged basic
molecules on cell surface
• Acidic dye – negatively charged
chromophore
• Repelled by cell surface
• Used to stain background
Nigrosin – acidic
• Negative stain.
 Preparing a 1. Preparing a smear

specimen for
microscopic Spread culture in thin Dry in air
examination film over slide

2. Heat fixing and staining

Pass slide through Flood slide with stain;

flame to heat fix rinse and dry

3. Microscopy

100×
Slide Oil
Figure 1.17
Place drop of oil on slide;
examine with 100× objective
lens
 Differential stains: the Gram stain

• Separates bacteria into 2

groups based on cell wall
structure
Gram positive – cells that
retain a primary stain
• Purple
Gram negative – cells that
lose the primary stain
• Take color of counterstain
• Red or pink.

Figure 1.18 a
Differential stains: the Gram stain

Purple cells are: Gram________

Pink cells are: Gram _________

Figure 1.18 b
 Other differential stains:
• Acid fast stain
• Detects mycolic acid in the cell wall of
the genus Mycobacterium

• Mycobacterium – retains primary stain

• Fuchsia (pink)
• Anything else on slide – color of
counterstain
• Blue
Endospore stain
• Endospores retain primary
• Green
• Cells counterstained
• Pink
Ex. Bacillus anthracis spores.
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 Improving Contrast in Light Microscopy

• Phase-contrast microscopy
• Phase ring amplifies differences in the refractive index of cell
and surroundings
• Improves the contrast of a sample without the use of a stain
• Allows for the visualization of live samples
• Resulting image is dark cells on a light background (Figure
1.19 b)
Improving Contrast in Light Microscopy
Dark field microscopy
• Specimen is illuminated with a hollow cone
of light (Fig. 1.19 c)

• Only refracted light enters the objective

• Specimen appears as a bright object on a

dark background

• Used to observe bacteria that don’t stain

well
• ex. Treponema pallidum – the causative
agent of syphilis.
 Improving Contrast in Light Microscopy
Fluorescence microscopy
• Used to visualize specimens that fluoresce
• Emit light of one color when illuminated with
another color of light
Cells may fluoresce naturally
ex. Photosynthetic Cyanobacteria have
chlorophyll
• Absorbs light at 430 nm (blue-violet)
• Emits at 670 nm (red)
Or after staining with Fluorescent dye
• ex. DAPI specifically binds to DNA.

Figure 1.2
Imaging Cells in Three Dimensions
• Differential interference contrast (DIC) microscopy
• Uses a polarizer to create two distinct beams of polarized light
• The two beams pass through the specimen and enter the objective
lens, where they recombine into one

• Gives structures such as endospores, vacuoles, and granules a

three-dimensional appearance (Figure 1.21)
• Structures not visible by bright-field microscopy are sometimes
visible by DIC Nucleus

Figure 1.21
 Imaging Cells in Three Dimensions
• Confocal scanning laser microscopy
(CSLM)
• Uses a computerized fluorescent
microscope coupled with a laser source
to generate a three-dimensional image
(Figure 1.22)
• Computer can focus the laser on single
layers of the specimen
• Different layers can then be compiled for
a three-dimensional image
• Resolution is 0.1 μm for CSLM Figure 1.22
 Probing Cell Structure: Electron Microscopy
• Electron microscopes use electrons
instead of photons to image cells and Electron
source
structures (Figure 1.23)

• Wavelength of electrons is much Evacuated

chamber
shorter than light  higher
Sample
port
resolution
• Two types of electron microscopes:
• Transmission electron Viewing
screen
microscopes (TEM)
• Scanning electron microscopes
(SEM) Figure 1.23
 Transmission electron microscope (TEM)
• Electron beam focused on
specimen by a condenser
• magnets used as lenses
• Electrons that pass through the
specimen are focused by two
sets of lenses
• compound microscope
• Electrons strike a fluorescent
viewing screen.

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 Transmission electron microscopy (TEM)
• High magnification and resolution (0.2 nm)
• Specimen must be very thin (20 – 60 nm)
• Unstained cells do a poor job of scattering electrons
• Must be stained with metals  lead or uranium
• Bind to cell structures to make them more electron dense
• Enables visualization of structures at molecular level

Figure 1.24 a
Difference in Resolution Between Light and
Transmission Electron Microscope

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Probing Cell Structure: Electron Microscopy
• Scanning electron microscopy (SEM)
• Specimen is coated with a thin film of heavy metal (e.g., gold)
• An electron beam scans the object
• Scattered electrons are collected by a detector, and an image is
produced (Figure 1.24 c)
• Allows an accurate 3D image of specimen’s surface.

SEM of
bacteria cells

Figure 1.24c
Activity 3: Mini open book quiz
1. Electron microscopy has greater ________ than light
microscopy, because the wavelength of visible light is
much larger than the wavelength of electrons.

A) contrast
B) magnification
C) resolution
D) penetration

2. Which of the following types of microscopy is especially

effective for viewing details of internal structures within live
cells?

A) phase-contrast microscopy
B) transmission electron microscopy
C) bright-field microscopy
D) scanning electron microscopy
3. Which of the following types of microscopy could be
used to visualize the layers of the cell membrane and the
cell wall?

A) phase-contrast microscopy
B) transmission electron microscopy
C) bright-field microscopy
D) confocal microscopy

4. True or False: Magnification is the ability to distinguish

between two objects as distinct and separate while resolution is
the optical enlargement of the object.
5. Bacterial cells are stained with cationic dyes to
increase their contrast so that:

A) They are visible by the human eye.

B) The cells are more easily seen against the
background.
C) No particular reason
D) Structures become visible.
7. How is confocal scanning laser microscopy (CSLM)
different from fluorescent microscopy?

A) CSLM uses a laser to emit electrons through a thick sample instead

of using light from a single color.

B) There is no difference between the two.

C) CSLM uses filters so that layers of cells can be distinguished,

fluorescent microscopy does not.

D) CSLM uses a laser to cause multiple planes of focus throughout the

specimen to fluoresce while fluorescence microscopes apply specific
wavelengths of light to the specimen, which then fluoresces a different
color based on its own natural pigments or on previous application of
fluorescent stains.
8. True or False. The major difference between electron
microscopes and light microscopes is that light microscopes
use a beam of light and objective lenses to view the specimen,
while electron microscopes use an electron beam in a vacuum
and electromagnets as lenses to visualize the specimen.
Chapter 2: Microbial Cell Structure and Function
II. Cells of Bacteria and Archaea - overview
Prokaryote (before nucleus)
• No membrane bound nucleus or organelles
• Generally smaller than eukaryotes
• Simple internal structure
• Divide by binary fission
• Most are unicellular

Bacteria (Eubacteria) Archaea (Archaebacteria)

• Diverse metabolism • Diverse metabolism
• Live in a broad range of • Live in extreme
ecosystems environments
• Pathogens and non- • Non-pathogens
pathogens
II. Cells of Bacteria and Archaea

• Cell Morphology
• Cell Size and the Significance of Being Small
Cell Morphology = cell shape
Coccus (pl. cocci)
• Roughly spherical
• ex. Streptococcus pyogenes

Bacillus (pl. bacilli)

• Rod shaped
• ex. E. coli

Spirillum (pl. spirilla)

• Spiral shaped
• ex. Spirillum volutans
Figure 2.1
 Cell Morphology = cell shape
• Cells with unusual shapes

Spirochete
ex. Treponema pallidum

Budding & appendaged bacteria

ex. Caulobacter crescentus Stalk
Hypha

Filamentous bacteria
ex. Streptomyces griseus

Figure 2.1
Cell Morphology
• Morphology typically does not predict physiology,
ecology, phylogeny, etc. of a prokaryotic cell
• May be selective forces involved in setting the
morphology
• Optimization for nutrient uptake (small cells and those
with high surface-to-volume ratio)
• Swimming motility in viscous environments or near
surfaces (helical or spiral-shaped cells)
• Gliding motility (filamentous bacteria)
Cell Size and the Significance of Being Small

• Size range for prokaryotes: 0.2 µm to >700 µm in

diameter
• Size range for eukaryotic cells: 10 to >200 µm in
diameter
• Despite the overlap in size, it can be said that
prokaryotes are very small compared with
eukaryotes
Prokaryote cell size
Average:
• E. coli ~ 1.0 x 3.0 mm
• Staphylococcus aureus ~ 1.0 mm diameter

Very small:
• Mycoplasma pneumoniae ~ 0.2 mm

Very large:
• Epulopiscium fishelsonii ~ 80 x 600
mm.
Figure 2.12
 Cell Size and the Significance of Being Small

• Surface-to-volume ratios, growth rates, and

evolution
• Advantages to being small (Figure 2.13)
• Small cells have more surface area
relative to cell volume than large
cells (i.e., higher S/V)
• Support greater nutrient
exchange per unit cell
volume
• Tend to grow faster than
larger cells
Figure 2.13
Cell Size and the Significance of Being Small

• Lower limits of cell size

• Cellular organisms <0.15 µm in diameter are unlikely
• Open oceans tend to contain small cells (0.2–0.4 µm in
diameter)
• Many pathogenic bacteria are also small (missing many
genes whose functions are supplied to them by host)