Bio260 Microbiology
Instructors:
Traci Kinkel, Ph.D
What is microbiology?
• The scientific discipline which studies
microbes or microorganisms
– Biology of microbes
– The interaction of microbes with other microbes,
the environment, and humans
The Microbial World
 All living things can be classified into one of
three groups, or domains
• Bacteria
• Archaea
• Eucarya
 Organisms in each domain share certain
important properties
Major Groups of Microbial World
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Microbial World
Infectious agents
(non-living)
Organisms
(living)
Domain
Bacteria
Archaea
Viruses
Eucarya
Eukaryotes
Prokaryotes (unicellular)
Algae
(unicellular or
multicellular)
Protozoa
(unicellular)
Protists
Fungi
(unicellular or
multicellular)
Helminths
(multicellular
parasites)
Viroids
Prions
Types of Microbes
1. Algae
2. Fungi
3. Protozoa
4. Bacteria
5. Viruses
• Which microbes are eukaryotes?
• Which are prokaryotes?
• Which can perform
photosynthesis?
• Which are classified based on
locomotion?
• Which have cell walls?
• Which have some type of
nucleic acid?
Domain Bacteria
 Bacteria
•
•
•
•
•
•
•
Single-celled prokaryotes
Prokaryote = “prenucleus”
No membrane-bound nucleus
No other membrane-bound organelles
DNA in nucleoid
Most have specific shapes (rod, spherical, spiral)
Rigid cell wall contains peptidoglycan (unique to
bacteria)
• Multiply via binary fission
• Many move using flagella
Domain Archaea
 Archaea
•
•
•
•
•
Like Bacteria, Archaea are prokaryotic
Similar shapes, sizes, and appearances to Bacteria
Multiply via binary fission
May move via flagella
Rigid cell walls
 However, major differences in chemical
composition
• Cell walls lack peptidoglycan
• Ribosomal RNA sequences different
 Many are extremophiles
• High salt concentration, temperature
Domain Eucarya
 Eucarya
•
•
•
•
Eukaryotes = “true nucleus”
Membrane-bound nucleus and other organelles
More complex than prokaryotes
Microbial members include fungi, algae, protozoa
• Algae and protozoa also termed protists
• Some multicellular parasites including helminths
(roundworms, tapeworms) considered as well
Domain Eucarya
 Algae
• Diverse group
• Single-celled or multicellular
• Photosynthetic
• Contain chloroplasts with
chlorophyll or other pigments
• Primarily live in water
• Rigid cell walls
• Many have flagella
• Cell walls, flagella distinct
from those of prokaryotes
Domain Eucarya
 Fungi
• Diverse group
• Single-celled (e.g., yeasts) or multicellular (e.g., molds,
mushrooms)
• Energy from degradation of organic materials
• Primarily live on land
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Reproductive
structures
(spores)
Mycelium
(a)
10 µm
(b)
a: © CDC/Janice Haney Carr; b: © Dr. Richard Kessel & Dr. Gene Shih/Visuals Unlimited
10 µm
Domain Eucarya
 Protozoa
•
•
•
•
•
•
Diverse group
Single-celled
Complex, larger than prokaryotes
Most ingest organic compounds
No rigid cell wall
Most motile
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20 µm
© Manfred Kage/Peter Arnold
Nomenclature
 Binomial System of Nomenclature: two words
•
•
•
•
•
Genus (capitalized)
Specific epithet, or species name (not capitalized)
Genus and species always italicized or underlined
E.g., Escherichia coli
May be abbreviated (e.g., E. coli)
1.4. Non-Living Members of the Microbial World




Viruses, viroids, prions
Acellular infectious agents
Not alive
Not microorganisms, so general term microbe
often used to include
1.4. Non-Living Members of the Microbial World
 Viruses
•
•
•
•
•
•
Nucleic acid packaged in protein coat
Variety of shapes
Infect living cells, termed hosts
Multiply using host machinery, nutrients
Inactive outside of hosts: obligate intracellular parasites
All forms of life can be infected by different types
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Nucleic acid
Protein coat
(a)
50 nm
(b) Protein coat
Nucleic acid
Tail
50 nm
(c)
Nucleic acid
a: © K.G. Murti/Visuals Unlimited; b: © Thomas Broker/Phototake; c: © K.G. Murti/Visuals Unlimited
50 nm
1.4. Non-Living Members of the Microbial World
 Viroids
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• Simpler than viruses
• Require host cell for replication
• Consist of single short piece of
RNA
• No protective protein coat
• Cause plant diseases
• Some scientists speculate they
may cause diseases in humans
PSTV
• No evidence yet
T7 DNA
PSTV
1 um
U.S. Department of Agriculture/Dr. Diemer
1.4. Non-Living Members of the Microbial World
 Prions
• Infectious proteins: misfolded versions of normal
cellular proteins found in brain
• Misfolded version forces normal version to misfold
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• Abnormal proteins bind to form fibrils
• Cells unable to function
• Cause several neurodegenerative
diseases in humans, animals
• Resistant to standard sterilization
procedures
50 nm
© Stanley B. Prusiner/Visuals Unlimited
Bacteria That Lack a Cell Wall
 Some bacteria lack a cell wall
• Mycoplasma species have extremely variable shape
• Penicillin, lysozyme do not affect
• Cytoplasmic membrane contains sterols that increase
strength
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2m
Courtesy of Dr. Edwin S. Boatman
Cell Walls of the Domain Archaea
 Members of Archaea have variety of cell walls
• Probably due to wide range of environments
• Includes extreme environments
•
•
•
•
However, Archaea less well studied than Bacteria
No peptidoglycan
Some have similar molecule pseudopeptidoglycan
Many have S-layers that self-assemble
• Built from sheets of flat protein or glycoprotein subunits
Major Groups of Microbial World
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Microbial World
Infectious agents
(non-living)
Organisms
(living)
Domain
Bacteria
Archaea
Viruses
Eucarya
Eukaryotes
Prokaryotes (unicellular)
Algae
(unicellular or
multicellular)
Protozoa
(unicellular)
Protists
Fungi
(unicellular or
multicellular)
Helminths
(multicellular
parasites)
Viroids
Prions
History of Microbiology
• It all started with the microscope!
– Zacharis Janssen (1600)
– Antoni van Leewenhoek (1632-1723)
– Robert Hooke (1665)
Where do cells come from?
• Spontaneous generation
– Francesco Redi (1668)
– John Needham (1745)
– Lazzaro Spallanzani (1765)
– Louis Pasteur (1861)
• Biogenesis
– Rudolf Virchow (1858)
Pasteur’s flasks
John Tyndall questions Pasteur’s experiments
• Could not reproduce Pasteur’s results
• Found that there were heat resistant forms of
microbes
• Same year (1876) Ferdinand Cohn discovers heat
resistant forms of bacteria called endospores
• 1877 Robert Koch demonstrates that anthrax caused
by Bacillus anthracis
1.5. Size in the Microbial World
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Nucleus
Small
molecules
Atoms
Proteins
Viruses
Mitochondria
Prion fibril
Lipids
Ribosomes Smallest
bacteria
Most
bacteria
Most eukaryotic cells
Adult roundworm
Human height
Electron microscope
Light microscope
Unaided human eye
0.1 nm
1 nm
10 nm
100 nm
1 µm
10 µm
The basic unit of length is the meter (m), and all
other units are fractions of a meter.
nanometer (nm) = 10–9 meter = .000000001 meter
micrometer (µm) = 10–6 meter = .000001 meter
millimeter (mm) = 10–3 meter = .001 meter
1 meter = 39.4 inches
100 µm
1 mm
1 cm
0.1 m
These units of measurement correspond to units
in an older but still widely used convention.
1 angstrom (Å) = 10–10 meter
1 micron (µ) = 10–6 meter
1m
10 m
Principles of Light Microscopy
• Light passes through specimen and then series
of magnifying lenses
• Bright-field microscope is most common type
• Three key concepts
– Magnification: apparent increase in size
• Modern compound microscopes have two lens types:
objective and ocular
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• Magnification is product of
objective (4x, 10x, 40x, and
100x) and ocular lens (10x)
• Condenser lens (between
light source and specimen)
focuses light on specimen,
does not magnify
Ocular lens
(eyepiece)
Magnifies the
image, usually
10-fold (10×).
Objective lens
A selection of lens
options provide
different
magnifications. The
total magnification is
the product of the
magnifying power of
the ocular lens and
the objective lens.
Specimen
stage
Condenser
Focuses
the light.
Light source
Iris diaphragm
Controls the
amount of light
that enters the
objective lens.
Rheostat
Controls the
brightness of the
light.
Courtesy of Leica, Inc., Deerfield, FL
Microscopy-Brightfield
Oil has same refractive index as glass
3.2. Microscopic Techniques: Dyes and
Staining
• Samples can be immobilized, stained to visualize
• Basic dyes (positive charge)
– Attracted to negatively charged cellular components
• Acidic dyes (negative charge)
– Negative staining: cells repel, so colors background
– Can be done as wet mount
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Spread thin film of
specimen over slide.
Allow to air dry.
Pass slide
through
flame to
heat-fix
specimen.
Flood the smear with
stain, rinse, and dry.
Examine with microscope.
3.3. Morphology of Prokaryotic Cells
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• Two types most common
Coccus
Rod (bacillus)
– Coccus: spherical
– Rod: cylindrical
• Variety of other shapes
(a)
– Vibrio, spirillum, spirochete
– Pleomorphic (many shapes)
– Great diversity often found in low
nutrient environments
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1 µm
Vibrio
(c)
(b)
11.4 µm
Spirillum
15 µm
(d)
15 µm
Spirochete
(a)
1 m
(b)
1 m
a: Courtesy of Walther Stoeckenius; b: Courtesy of James T. Staley
(e)
7.5 µm
(a): © SciMAT/Photo Researchers, Inc.; (b, c, d, e): © Dennis Kunkel Microscopy inc.
Groupings
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• Most prokaryotes divide
by binary fission
– Cells often stick together
following division
– Form characteristic
groupings
Chains
Diplococcus
Cell divides
in one plane.
Chain of cocci
(a)
Packets
Cell divides
in two or more planes
perpendicular to one
another.
Packet
(b)
Clusters
Cell divides
in several planes at
random.
Cluster
(c)
(a): (top): © George Musil/Visuals Unlimited; (bottom): © David M. Phillips/Visuals Unlimited; (b): © R. Kessel & C.
Shih/Visuals Unlimited; (c): © Oliver Mecks/Photo Researchers, Inc.
The Prokaryotic Cell
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Pilus
Ribosomes
Cytoplasm
Chromosome
(DNA)
Nucleoid
Cell wall
Flagellum
(b)
Capsule
Cell wall
Cytoplasmic
membrane
(a)
(b): Courtesy of L. Santo, H. Hohl, and H. Frank, "Ultrastructure of Putrefactive Anaerobe 3679h During Sporulation,“
Journal of Bacteriology 99:824, 1969. American Society for Microbiology
0.5 µm
Permeability of Lipid Bilayer (Cell Membrane)
• Cytoplasmic membrane is selectively permeable
– O2, CO2, N2, small hydrophobic molecules, and water
pass freely
– Some cells facilitate water passage with aquaporins
– Other molecules must be moved across membrane via
transport systems
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Pass through easily: Passes through:
Gases (O2, CO2, N2) Water
Small hydrophobic
molecules
Do not pass through:
Sugars
Ions
Amino acids
ATP
Macromolecules
Water
Aquaporin
(a) The cytoplasmic membrane is selectively permeable. Gases, small
hydrophobic molecules, and water are the only substances that
pass freely through the phospholipid bilayer.
(b) Aquaporins allow water to pass through the cytoplasmic membrane
more easily.
Permeability of Lipid Bilayer
• Simple Diffusion
– Movement from high to
low concentration
– Speed depends on
concentration
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Water flows across a
membrane toward the
hypertonic solution.
Hypotonic solution
Hypertonic solution
Water flow
Solute
molecule
• Osmosis
– Diffusion of water across
selectively permeable
membrane due to
unequal solute
concentrations
Water flow
Water flows in
Water flows out
• Three terms:
– Hypertonic
– Isotonic
– Hypotonic
Cytoplasmic membrane is
forced against cell wall.
Cytoplasmic membrane
pulls away from cell wall.
Cytoplasmic Membrane and Energy Transformation
• Electron Transport Chain embedded in membrane
– Critical role in converting energy into ATP
• Eukaryotes use membrane-bound organelles
– Use energy from electrons to move protons out of cell
– Creates electrochemical gradient across membrane
• Energy called proton motive force
• Harvested to drive
cellular processes
including ATP
H+
H+
synthesis and
H+
H+
some forms of
transport, motility
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H+
H+
H+
H+
H+
H+
H+
H+
H+
Electron transport chain
OH–
OH–
H+
OH– OH–
OH– OH–
–
OH– OH
H+
OH–
3.5. Directed Movement of Molecules Across
Cytoplasmic Membrane
• Facilitated diffusion is a form of passive transport
– Movement down gradient; no energy required
• Not typically useful in low-nutrient environments
• Active transport requires energy
– Movement against gradient
– Two main mechanisms
• Use proton motive force
• Use ATP (ABC transporter)
• Group Translocation
– Chemically alter compound
• Phosphorylation common
– Glucose, for example
3.5. Directed Movement of Molecules Across
Cytoplasmic Membrane
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Transported
substance
Binding
protein
H+
H+
H+
H+
H+
(a) Facilitated diffusion
Transporter allows a substance
to move across the membrane,
but only down the concentration
gradient.
(b) Active transport, using
proton motive force as an
energy source.
P P P
ATP
P P
ADP
R
P
+ Pi
Active transport, using ATP as an
energy source. A binding protein
gathers the transported molecules.
Transporter uses energy (ATP or proton motive force) to move a
substance across the membrane and against a concentration
gradient.
R
P
P
(c) Group translocation
Transporter chemically
alters the substance
as it is transported
across the membrane.
P
Flagella - motility
• Rotate like a propeller
• Proton motive force used for
energy
• Presence/arrangement can
be used as an identifying
marker
E. coli O157:H7
• Peritrichous
• Polar
• Other (ex. tuft on both ends)
3.6. Cell Wall
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
N-acetylmuramic acid
(NAM)
– Alternating series of
subunits form glycan
chains
• N-acetylmuramic acid
(NAM)
• N-acetylglucosamine
(NAG)
CH2OH
CH2OH
O
H
O
• Cell wall is made
from peptidoglycan
N-acetylglucosamine
(NAG)
O
H
O
HC
C
O
H
H
NH
C
CH3
O
H
OH
H
H
NH
O
O
H
C
CH3
Chemical structure
O
CH3
OH
NAG
NAM
NAG
NAM
Glycan
chain
Peptidoglycan
Peptide interbridge
(Gram-positive cells)
Tetrapeptide
chain
(amino acids)
Glycan chains
- NAG and NAM
NAM
– Tetrapeptide chain
NAG
NAM
NAM
NAG
NAG
Glycan
chain
(string of four amino acids)
links glycan chains
Interconnected glycan chains
form a large sheet. Multiple
connected layers create a
three-dimensional molecule.
Tetrapeptide chain
(amino acids)
Peptide interbridge
The Gram-Positive Cell Wall
• Gram-positive cell wall has thick peptidoglycan layer
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
N-acetylglucosamine
N-acetylmuramic acid
Teichoic acid
Peptidoglycan
and teichoic acids
Gel-like
material
Peptidoglycan
(cell wall)
Cytoplasmic
membrane
Gel-like
material
Gram-positive
(b)
Cytoplasmic
membrane
Peptidoglycan
Cytoplasmic
membrane
(a)
(c)
(c): © Terry Beveridge, University of Guelph
0.15 µm
The Gram-Negative Cell Wall
• Gram-negative
cell wall has
thin peptidoglycan layer
• Outside is
unique outer
membrane
• Periplasm
• LPS
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O antigen
(varies in length and
composition)
Porin protein
Core polysaccharide
Lipid A
Lipopolysaccharide
(LPS)
(b)
Outer
membrane
(lipid bilayer)
Outer
membrane
Peptidoglycan
Lipoprotein
Periplasm
Cytoplasmic
membrane
Peptidoglycan
Periplasm
(c)
Cytoplasmic
membrane
(inner membrane;
lipid bilayer)
Outer
Cytoplasmic
Peptidoglycan membrane Periplasm membrane
(a)
(d)
(d): © Terry Beveridge, University of Guelph
0.15 µm
3.9. Internal Structures
• Chromosome forms gel-like region: the
nucleoid
– Single circular double-stranded DNA
• Packed tightly via binding proteins and supercoiling
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
DNA
(a)
0.5 µm
(b)
(a): © CNRI/SPL/Photo Researchers, Inc.; (b): © Dr. Gopal Murti/SPL/Photo Researchers
1.3 µm
3.9. Internal Structures
• Ribosomes are involved in
protein synthesis
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
– Facilitate joining of amino acids
– Prokaryotic ribosomes are
70S
50S subunit
30S subunit
• Made from 30S and 50S
– Eukaryotic ribosomes are 80S
• Important medically:
antibiotics
impacting 70S ribosome do
not
affect 80S ribosome
30S + 50S
combined
70S ribosome
Internal Structures:
Endospores
The Eukaryotic Cell
Comparisons of Eukaryotic and Prokaryotic Cells