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10/5/2013
LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Chapter 2
Lectures by
John Zamora
Middle Tennessee State University
A Brief Journey to
the Microbial World
© 2012 Pearson Education, Inc.
I. Seeing the Very Small
•
•
•
•
2.1 Some Principles of Light Microscopy
2.2 Improving Contrast in Light Microscopy
2.3 Imaging Cells in Three Dimensions
2.4 Electron Microscopy
© 2012 Pearson Education, Inc.
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2.1 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
Animation: Light Microscopy
© 2012 Pearson Education, Inc.
2.1 Some Principles of Light Microscopy
• Bright-field scope (Figure 2.1a)
– Specimens are visualized because of differences
in contrast (density) between specimen and
surroundings (Figure 2.2)
• Two sets of lenses form the image (Figure 2.1b)
– Objective lens and ocular lens
– Total magnification = objective magnification 
ocular magnification
– Maximum magnification is ~2,000
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Figure 2.1a A light microscope
Ocular
lenses
Specimen on
glass slide
Objective lens
Stage
Condenser
Focusing knobs
Light source
© 2012 Pearson Education, Inc.
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Figure 2.2 Bright-field photomicrographs of pigmented microorganisms
© 2012 Pearson Education, Inc.
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Figure 2.1b Path of light through a compound light microscope. Besides 10x, ocular lenses are available in
15–30x magnifications
Magnification Light path
100, 400,
1000
10
Visualized
image
Eye
Ocular lens
Intermediate image
(inverted from that
of the specimen)
10, 40, or
100 (oil)
Objective lens
Specimen
None
Condenser lens
Light source
© 2012 Pearson Education, Inc.
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2.1 Some Principles of Light Microscopy
• Resolution: the ability to distinguish two adjacent
objects as separate and distinct
– Resolution is determined by the wavelength of
light used and numerical aperture of lens
– Limit of resolution for light microscope is about
0.2  m
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2.2 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
Animation: Microscopy & Staining Overview
Animation: Staining
© 2012 Pearson Education, Inc.
Figure 2.3 Staining cells for microscopic observation
I. Preparing a smear
Spread culture in thin
film over slide
Dry in air
II. Heat fixing and staining
Pass slide through
flame to heat fix
Flood slide with stain;
rinse and dry
III. Microscopy
Slide
Oil
Place drop of oil on slide;
examine with 100
objective lens
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2.2 Improving Contrast in Light Microscopy
• Differential stains: the Gram stain
• Differential stains separate bacteria into groups
• The Gram stain is widely used in microbiology
(Figure 2.4a)
– Bacteria can be divided into two major groups:
gram-positive and gram-negative
– Gram-positive bacteria appear purple and gramnegative bacteria appear red after staining
(Figure 2.4b)
© 2012 Pearson Education, Inc.
Figure 2.4a The Gram stain - Steps in the procedure
Step 1
Flood the heat-fixed
smear with crystal
violet for 1 min
Result:
All cells purple
Step 2
Add iodine solution
for 1 min
Result:
All cells
remain purple
Step 3
Decolorize with
alcohol briefly
— about 20 sec
Result:
Gram-positive
cells are purple;
gram-negative
cells are colorless
Step 4
G-
Result:
Gram-positive
(G+) cells are purple;
gram-negative (G-) cells
are pink to red
© 2012 Pearson Education, Inc.
Counterstain with
safranin for 1–2 min
G+
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Figure 2.4b Microscopic observation of gram-positive (purple) and gram-negative (pink) bacteria
© 2012 Pearson Education, Inc.
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2.2 Improving Contrast in Light Microscopy
• Phase-Contrast Microscopy
– Invented in 1936 by Frits Zernike
– 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 2.5)
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2.2 Improving Contrast in Light Microscopy
• Dark-Field Microscopy
– Light reaches the specimen from the sides
– Light reaching the lens has been scattered by
specimen
– Image appears light on a dark background
(Figure 2.5)
– Excellent for observing motility
© 2012 Pearson Education, Inc.
Figure 2.5 Cells visualized by different types of light microscopy. The same field of cells of the baker’s yeast
Saccharomyces cerevisiae visualized by (a) bright-field microscopy, (b) phase-contrast microscopy, and (c)
dark-field microscopy. Cells average 8–10 µm wide.
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2.2 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 (Figure 2.6)
– Cells fluoresce naturally (autofluorescence) or
after they have been stained with a
fluorescent dye like DAPI
– Widely used in microbial ecology for
enumerating bacteria in natural samples
© 2012 Pearson Education, Inc.
Figure 2.6 Fluorescence microscopy. Same cells are observed by bright-field microscopy in part a and by
fluorescence microscopy in part b. Cells fluoresce red because they contain chlorophyll a and other pigments.
Fluorescence photomicrograph of cells of Escherichia coli made fluorescent by staining with the
fluorescent dye DAPI.
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2.3 Imaging Cells in Three Dimensions
• Confocal Scanning Laser Microscopy (CSLM)
– Uses a computerized microscope coupled with a
laser source to generate a three-dimensional
image (Figure 2.8)
– Computer can focus the laser on single layers of
the specimen
– Different layers can then be compiled for a threedimensional image
– Resolution is 0.1  m for CSLM
© 2012 Pearson Education, Inc.
Figure 2.8 Confocal scanning laser microscopy
(a) Confocal image
of a microbial biofilm
community cultivated in the
laboratory.
The green, rod-shaped cells
are Pseudomonas aeruginosa
experimentally introduced
into the biofilm.
Other cells of different colors
are present at different
depths in the biofilm.
(b) Confocal image of a
filamentous cyanobacterium
growing in a soda lake. Cells
are about 5 µm wide.
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2.4 Electron Microscopy
• Electron microscopes use electrons instead
of photons to image cells and structures
(Figure 2.9)
• Two types of electron microscopes:
– Transmission electron microscopes (TEM)
– Scanning electron microscopes (SEM)
© 2012 Pearson Education, Inc.
Figure 2.9 The electron microscope
Electron
source
Evacuated
chamber
Sample
port
Viewing
screen
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2.4 Electron Microscopy
• Transmission Electron Microscopy (TEM)
–
–
–
–
Electromagnets function as lenses
System operates in a vacuum
High magnification and resolution (0.2 nm)
Enables visualization of structures at the
molecular level (Figure 2.10a and b)
– Specimen must be very thin (20–60 nm) and
be stained
Animation: Electron Microscopy
© 2012 Pearson Education, Inc.
Figure 2.10a Electron micrographs. (a) Micrograph of a thin section of a dividing bacterial cell, taken by
transmission electron microscopy (TEM). Note the DNA forming the nucleoid. The cell is about 0.8 µm wide.
Cytoplasmic
membrane
© 2012 Pearson Education, Inc.
Septum Cell wall
DNA
(nucleoid)
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2.4 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 2.10c)
– Even very large specimens can be observed
– Magnification range of 15–100,000
© 2012 Pearson Education, Inc.
Figure 2.10c Scanning electron micrograph of bacterial cells. A single cell is about 0.75 µm wide.
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II. Cell Structure and Evolutionary History
• 2.5 Elements of Microbial Structure
• 2.6 Arrangement of DNA in Microbial Cells
• 2.7 The Evolutionary Tree of Life
© 2012 Pearson Education, Inc.
2.5 Elements of Microbial Structure
• All cells have the following in common:
– Cytoplasmic membrane
– Cytoplasm
– Ribosomes
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2.5 Elements of Microbial Structure
• Eukaryotic vs. Prokaryotic Cells
– Eukaryotes (Figures 2.11b and 2.12c)
• DNA enclosed in a membrane-bound nucleus
• Cells are generally larger and more complex
• Contain organelles
– Prokaryotes (Figures 2.11a and 2.12a and b)
• No membrane-enclosed organelles, no nucleus
• Generally smaller than eukaryotic cells
© 2012 Pearson Education, Inc.
Figure 2.11b Internal structure of eukaryotic cells
Cytoplasmic
membrane
Endoplasmic
reticulum
Ribosomes
Nucleus
Nucleolus
Nuclear
membrane
Golgi
complex
Cytoplasm
Mitochondrion
Chloroplast
Eukaryote
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Figure 2.12c Electron micrograph of sectioned eukaryotic cells
Eukaryote
Cytoplasmic
membrane
Nucleus
Cell
wall
Mitochondrion
Eukarya
© 2012 Pearson Education, Inc.
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Figure 2.11a Internal structure of prokaryotic cells
Cytoplasm
Nucleoid
Ribosomes
Plasmid
Cell wall
Prokaryote
© 2012 Pearson Education, Inc.
Cytoplasmic
membrane
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Figure 2.12a Electron micrograph of sectioned prokaryotic cells
Prokaryotes
(a) Bacteria
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Prokaryotes
Archaea
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2.5 Elements of Microbial Structure
• Viruses
– Not considered cells
– No metabolic abilities of their own
– Rely completely on biosynthetic machinery of
infected cell
– Infect all types of cells
– Smallest virus is 10 nm in diameter
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2.6 Arrangement of DNA in Microbial Cells
• Genome
– A cell’s full complement of genes
• Prokaryotic cells generally have a single, circular
DNA molecule called a chromosome
– DNA aggregates to form the nucleoid region
(Figure 2.14)
– Prokaryotes also may have small amounts of
extra-chromosomal DNA called plasmids that
confer special properties (e.g., antibiotic
resistance)
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Figure 2.14 The nucleoid
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2.6 Arrangement of DNA in Microbial Cells
• Eukaryotic DNA is linear and found within the
nucleus
– Associated with proteins that help in folding of
the DNA
– Usually more than one chromosome
– Typically two copies of each chromosome
– During cell division, nucleus divides by mitosis
– During sexual reproduction, the genome is
halved by meiosis
© 2012 Pearson Education, Inc.
2.6 Arrangement of DNA in Microbial Cells
• Escherichia coli Genome
–
–
–
–
4.64 million base pairs
4,300 genes
1,900 different kinds of protein
2.4 million protein molecules
• Human Cell
– 1,000 more DNA per cell than E. coli
– 7 more genes than E. coli
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2.7 The Evolutionary Tree of Life
• Evolution
– The process of change over time that results in
new varieties and species of organisms
• Phylogeny
– Evolutionary relationships between organisms
– Relationships can be deduced by comparing
genetic information in the different specimens
– Ribosomal RNA (rRNA) is excellent for
determining phylogeny
– Relationships visualized on a phylogenetic tree
(Figure 2.16)
© 2012 Pearson Education, Inc.
Figure 2.16 Ribosomal RNA (rRNA) gene sequencing and phylogeny. (a) DNA is extracted from cells. (b)
Many identical copies of a gene encoding rRNA are made by the polymerase chain reaction
DNA
DNA sequencing
Aligned rRNA
gene sequences
Gene encoding
ribosomal RNA
Cells
Isolate
DNA
© 2012 Pearson Education, Inc.
PCR
Sequence
analysis
Generate
phylogenetic
tree
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2.7 The Evolutionary Tree of Life
• Comparative rRNA sequencing has defined three
distinct lineages of cells called domains:
– Bacteria (prokaryotic)
– Archaea (prokaryotic)
– Eukarya (eukaryotic)
• Archaea and Bacteria are NOT closely related
(Figure 2.17)
• Archaea are more closely related to Eukarya than
Bacteria
• Eukaryotic microorganisms were the ancestors of
multicellular organisms (Figure 2.17)
© 2012 Pearson Education, Inc.
Figure 2.17 The phylogenetic tree of life as defined by comparative rRNA gene sequencing
BACTERIA
ARCHAEA
EUKARYA
Animals
Entamoebae
Green nonsulfur
bacteria
Mitochondrion
GramProteobacteria positive
bacteria
Chloroplast
Euryarchaeota
Methanosarcina
MethanoExtreme
Crenarchaetoa
bacterium
halophiles
Thermoproteus
Methanococcus
Thermoplasma
Pyrodictium
Fungi
Plants
Ciliates
Thermococcus
Cyanobacteria
Flavobacteria
Slime
molds
Marine
Crenarchaeota
Pyrolobus
Flagellates
Methanopyrus
Trichomonads
Thermotoga
Microsporidia
Thermodesulfobacterium
Aquifex
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LUCA
Diplomonads
(Giardia)
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III. Microbial Diversity
•
•
•
•
2.8 Metabolic Diversity
2.9 Bacteria
2.10 Archaea
2.11 Phylogenetic Analyses of Natural
Microbial Communities
• 2.12 Microbial Eukarya
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2.8 Metabolic Diversity
• The diversity in microbial cells is the product
of almost 4 billion years of evolution
• Microorganisms differ in size, shape, motility,
physiology, pathogenicity, etc.
• Microorganisms have exploited every
conceivable means of obtaining energy from
the environment
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2.8 Metabolic Diversity
• Chemoorganotrophs
– Obtain their energy from the oxidation of organic
molecules (Figure 2.18)
– Aerobes use oxygen to obtain energy
– Anaerobes obtain energy in the absence of
oxygen
• Chemolithotrophs
– Obtain their energy from the oxidation of
inorganic molecules (Figure 2.18)
– Process found only in prokaryotes
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2.8 Metabolic Diversity
• Phototrophs
– Contain pigments that allow them to use light
as an energy source (Figure 2.18)
– Oxygenic photosynthesis produces oxygen
– Anoxygenic photosynthesis does not produce
oxygen
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Figure 2.18 Metabolic options for conserving energy
Energy Sources
Chemicals
Light
Chemotrophy
Phototrophy
Organic
chemicals
Inorganic
chemicals
(glucose, acetate, etc.)
(H2, H2S, Fe2+, NH4+, etc.)
Chemoorganotrophs Chemolithotrophs Phototrophs
(glucose  O2
CO2  H2O)
© 2012 Pearson Education, Inc.
(H2  O2
H2O)
(light)
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2.8 Metabolic Diversity
• All cells require carbon as a major nutrient
– Autotrophs
• Use carbon dioxide as their carbon source
• Sometimes referred to as primary producers
– Heterotrophs
• Require one or more organic molecules for
their carbon source
• Feed directly on autotrophs or live off
products produced by autotrophs
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2.8 Metabolic Diversity
• Organisms that inhabit extreme environments
are called extremophiles
• Habitats include boiling hot springs, glaciers,
extremely salty bodies of water, and high-pH
environments
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2.9 Bacteria
• The domain Bacteria contains an enormous
variety of prokaryotes (Figure 2.19)
• All known pathogenic prokaryotes are Bacteria
• The Proteobacteria make up the largest phylum of
Bacteria
– Gram-negative
• Examples: E. coli, Pseudomonas, and Salmonella
• Gram-positive phylum united by phylogeny and
cell wall structure
– Cyanobacteria are relatives of gram-positive
bacteria
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Figure 2.19 Phylogenetic tree of some representative Bacteria
Spirochetes
Deinococcus
Green sulfur Planctomyces
bacteria
Green nonsulfur
bacteria
Chlamydia
Cyanobacteria
Thermotoga
Gram-positive
bacteria
OP2
Aquifex
Proteobacteria
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2.9 Bacteria
• Many Other Phyla of Bacteria
– Green sulfur bacteria and green nonsulfur
bacteria are photosynthetic
– Deinococcus is extremely resistant to
radioactivity
– Chlamydia are obligate intracelluar parasites
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2.10 Archaea
• Two Phyla of the Domain Archaea
– Euryarchaeota (Figure 2.28)
• Methanogens: degrade organic matter
anaerobically, produce methane (natural
gas)
• Extreme halophiles: require high salt
concentrations for metabolism and
reproduction
• Thermoacidophiles: grow in moderately high
temperatures and low-pH environments
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2.10 Archaea
– Crenarchaeota (Figure 2.28)
• Vast majority of cultured Crenarchaeota are
hyperthermophiles
• Some live in marine, freshwater, and soil
systems
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Figure 2.28 Phylogenetic tree of some representative Bacteria
Marine group
Crenarchaeota
Halobacterium
Natronobacterium
Marine group
Euryarchaeota
Sulfolobus
Methanobacterium
Halophilic
methanogens
Methanocaldococcus
Thermoproteus
Pyrococcus
Methanosarcina
Thermoplasma
Pyrolobus
Methanopyrus
Desulfurococcus
Hyperthermophiles
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2.11 Phylogenetic Analyses of Natural
Microbial Communities
• Microbiologists believe that we have cultured
only a small fraction of the Archaea and
Bacteria
• Studies done using methods of molecular
microbial ecology, devised by Norman Pace
– Microbial diversity is much greater than
laboratory culturing can reveal
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2.12 Microbial Eukarya
• Eukaryotic microorganisms include algae, fungi,
protozoa, and slime molds (Figure 2.32)
– Protists include algae and protozoa
• The algae are phototrophic (Figure 2.33a)
• Protozoa NOT phototrophic (Figure 2.33c)
– Fungi are decomposers (Figure 2.33b)
– Algae and fungi have cell walls, whereas protozoa
and slime molds do not
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Figure 2.32 Phylogenetic tree of some representative Eukarya
Flagellates
Diplomonads
Trichomonads
Slime
molds
Ciliates
Animals
Green algae
Plants
Red algae
Fungi
Diatoms
Brown algae
Early-branching, lack mitochondria
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Figure 2.33a Microbial Eukarya - Algae
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Figure 2.33b Microbial Eukarya - Fungi
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Figure 2.33c Microbial Eukarya - Protozoa
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2.12 Microbial Eukarya
• Lichens are a mutualistic relationship
between two groups of protists (Figure 2.34)
– Fungi and cyanobacteria
– Fungi and algae
• Microbial eukaryotes are comprehensively
discussed in Chapter 20
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Figure 2.34 Lichens. (a) An orange-pigmented lichen growing on a rock, and (b) a yellow-pigmented lichen
growing on a dead tree stump,
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