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Summaries – 1
BI-311
Ch. 1
The Historical Roots of Microbiology: The Science
Ferdinand Cohn
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Founded the field of bacteriology
Recognized distinction between
prokaryotic and eukaryotic
cellular organization
Discovered bacterial endospores
The Historical Roots of Microbiology:
Louis Pasteur
• Discredited the theory of Spontaneous
Generation.
• Introduced control of microbial growth.
• Discovered lactic acid bacteria
• Role of yeast in alcohol fermentation
• Rabies vaccine
The Historical Roots of Microbiology:
Robert Koch
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Growth of pure cultures of microorganisms
Solid growth media
Discovered cause of tuberculosis.
Developed criteria for the study of infectious
microorganisms
• Kochst Postulates.
Koch’s Postulates
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OBSERVE: The presence of suspected
pathogenic microorganism correlates positively
with the symptoms of the diseased and negative
with healthy control
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ISOLATE the suspected pathogen into axenic
culture
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INFECT a healthy animal with cultured strain.
Observe whether the same symptoms show
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RE-ISOLATE the pathogen from the new
victim and compare both cultures
The Historical Roots of Microbiology: General Microbiology
- Microbial Ecology and Diversity
Martinus Beijerinck
• Enrichment Culture Technique
• Concept of Virus
Sergey Winogradsky
• Concept of Chemolithotrophy and
Autotrophy
Chapter 2
(in Brock Biology of Microorganisms 2012)
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Incident light microscopy (dissecting)
Transmitted light microscopy (compound)
Phase contrast
Dark field
Differential Interference Contrast (DIC)
Fluorescence microscopy
Confocal Scanning Light Microcopy (CSLM),
Transmission electron microscopy (TEM)
Scanning electron microscopy (SEM)
The atomic force microscope
Principles of Light Microscopy
• Bright-field scope
– Specimens are visualized because of differences in
contrast (density) between specimen and surroundings
• Two sets of lenses form the image
– Objective lens and ocular lens
– Total magnification = objective magnification  ocular
magnification
– Maximum magnification is ~2,000
• 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
Other microscope techniques
•Differential Interference Contrast (DIC) and
Confocal Scanning Light Microcopy (CSLM)
allow for greater three-dimensional imaging
than other forms of light microscopy,
• Confocal microscopy allows imaging through
thick specimens.
• The atomic force microscope yields a
detailed three-dimensional image of live
preparations.
Improving Contrast in Light Microscopy
• Improving contrast results in a better final image
• Staining improves contrast
– Positively charged dyes can be used to stain cells (bind to
negatively charged components such as nucleic acids,
acidic polysaccharides) to improve their contrast
– Dyes are organic compounds that bind to specific cellular
materials
– Examples of common stains are Methylene blue,
Safranin, Crystal violet
– Differential staining (Gram staining): Crystal violet and
Safranin to differentiate Gram(+)ve and (-)ve microbes
(Christian Gram-1984)
Improving Contrast in Light Microscopy
• Differential stains: the Gram stain
• The Gram stain is widely used in
microbiology to distinguish between
Bacteria with different cell wall structure:
Gram-positive bacteria appear purple and
gram-negative bacteria appear red after
staining and counterstaining
Gram
Staining
Step 1
Flood the heat-fixed
smear with crystal
violet for 1 min
Result:
All cells purple
Step 2
Unknown bacteria
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
Counterstain with
safranin for 1–2 min
G+
Gram Staining
Gram positive
(S. aureus)
Gram negative
(E. coli)
Gram positive
(Streptococcus)
Gram negative
(E. coli)
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
– Computer can focus the laser on single layers of the
specimen
– Cells are (i) either stained with fluorescent dyes, or (ii)
different layers in specimen are assigned colors to
generate false color images
– Different layers are then be compiled for a 3-D image
– Resolution is 0.1 m
– Applications: Thick biofilms, Microbial ecology
Electron microscopes
use electron beams instead of light. They have
far greater resolving power than do light
microscopes, the limits of resolution being about
0.2 nm. Two major types of electron microscopy
are performed:
Transmission Electron Microscopy (TEM), for
observing internal cell structure down to the
molecular level, and
Scanning Electron Microscopy (SEM), useful for
three-dimensional imaging and for examining
surfaces.
2.4 Electron Microscopy
• Transmission Electron Microscopy (TEM)
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Electromagnets function as lenses
System operates in a vacuum
High magnification and resolution (0.2 nm)
Enables visualization of structures at the
molecular level
– Specimen must be very thin (20–60 nm) and
be stained with compounds such as osmic
acid, permanganate, uranium, lanthanum or
lead salts (these contain atoms of high
Atomic weight, they scatter electrons well to
improve contrast)
Scanning Electron Microscopy – SEM
Glutaraldehyde-fixed, critical point-dried, goldpaladium coated
Elements of Microbial Structure
• Eukaryotic vs. Prokaryotic Cells
– Eukaryotes
• DNA enclosed in a membrane-bound nucleus
• Cells are generally larger and more complex (as
small at 0.8 m to several 100 m)
• Contain organelles
– Prokaryotes
• No membrane-enclosed organelles, no nucleus
• Generally smaller than eukaryotic cells
• Typical prokaryotic cell is ~1-5 m long, 1 m wide
Eukaryotic cell
Freeze-etched preparation
Carbon-coated,
Gold-shaded, TEM image
TEM’s of sectioned cells from each of the
domains of living organisms
Cytoplasmic
membrane
Cell wall
Nucleus
Mitochondrion
Gene, Genomes and Proteins
comparison
• Escherichia coli Genome
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~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
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) sequencing method is
excellent for determining phylogeny
– Relationships visualized on a phylogenetic
tree
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
• Archaea are more closely related to
Eukarya than Bacteria
Metabolic Diversity by Energy Source
• Chemoorganotrophs
– Obtain their energy from the oxidation of organic
molecules
– Aerobes use oxygen to obtain energy
– Anaerobes obtain energy in the absence of oxygen
• Chemolithotrophs
– Obtain their energy from the oxidation of inorganic
molecules
– Process found only in prokaryotes
• Phototrophs
– Contain pigments that allow them to use light as an
energy source
– Oxygenic photosynthesis produces oxygen
– Anoxygenic photosynthesis does not produce oxygen
Metabolic Diversity by C source
• All cells require carbon as a major nutrient
– Autotrophs
• Use CO2 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
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
(Metagenome?)
– More high-throughput techniques
Summary Microscopy
• Microscopes are essential for studying
microorganisms
• Inherent limit of bright field microscopy can
be overcome by use of stains, phase
contrast or dark-filed microcopy
• DIC and CFLM allows enhanced 3D
imaging
• AFM used for 3D imaging of live cells
• Electron microscopes have the best
resolving power
Summary Genes
• Genes govern the properties of a cell
• DNA is arranged in cells as chromosomes
• Prokaryotes (most) have single
chromosome
• Eukaryotes have multiple copies
• rRNA sequencing have defined 3 domains
of life
Summary Diversiy
• All cells need C and energy for growth
– Chemoorganotrophs: organic chemicals as energy source
– Chemolithotrophs: inorganic chemicals as energy source
– Phototrophs: Light as energy source
– Autotrophs: CO2 as C-source
– Heterotrophs: organic compounds as C-source
– Extremophiles: Can live in extreme environmental
conditions
• Bacterial Phyla: Proteobacteria, Gram positive
bacteria, Cyanobacteria, green bacteria
• Archaea: Euryarchaeota and Crenarchaeota
• Microbial Eukarya: Protists (algae and protozoa),
fungi and slime molds, Lichens
Cell Structure and Funtion
Chapter 3
• (in Brock Biology of Microorganisms 2012)
Macromolecules
• Organic chemistry = chemistry of carbon
• Biochemistry = chemistry of macromolecules
• Water = solvent & chemical bonding
properties: polarity, hidrophilic vs. hydrophobic
H-bonds, glycosidic, esteric, etheric, peptide.
• Biogenic elements = C, O, H, N, S, P construct
polymers from monomers: polysaccharides,
(phospho-)lipids, polypeptides, polynucleotides
CARBOXYL
ESTER
ETHER
ALDEHYDE
PHOSPHO-ESTER ACID ANHYDRIDE
ALCOHOL
THIOESTER
KETO
PHOSPHO ANHYDRIDE
• The cell walls of Bacteria contain a polysaccharide
called peptidoglycan.
• This material consists of strands of alternating
repeats of N-acetylglucosamine and Nacetylmuramic acid, with the latter cross-linked
between strands by short peptides. Many sheets of
peptidoglycan can be present, depending on the
organism.
• Archaea lack peptidoglycan but contain walls made
of other polysaccharides or of protein. The enzyme
lysozyme destroys peptidoglycan, leading to cell lysis
in Bacteria but not in Archaea
• In addition to peptidoglycan, gram-negative
Bacteria contain an outer membrane
consisting of lipopolysaccharide, protein, and
lipoprotein.
• Proteins called porins allow for permeability
across the outer membrane.
•The space between the membranes is the
periplasm, which contains various proteins
involved in important cellular functions.
Prokaryotic cells often contain various surface
structures. These include:
fimbriae
pili
S-layers
capsules
slime layers.
These structures have several functions,
but a key one is in attaching cells to a solid
surface.
Prokaryotic cells often contain internal
granules such as sulfur, PHB, polyphosphate,
PHAs, and magnetosomes. These substances
function as storage materials or in
magnetotaxis.
Gas vesicles are small gas-filled structures
made of protein that function to confer
buoyancy on cells. Gas vesicles contain two
different proteins arranged to form a gas
permeable, but watertight structure: Gas
Vesicle Proteins GVP-a and GVP-c.
The endospore is a highly resistant
differentiated bacterial cell produced by
certain gram-positive Bacteria.
• Endospore formation leads to a highly
dehydrated structure that contains essential
macromolecules and a variety of substances
such as calcium dipicolinate and small acidsoluble proteins, absent from vegetative cells.
• Endospores can remain dormant indefinitely
but germinate quickly when the appropriate
trigger is applied.
• Motility in most microorganisms is due to
flagella. In prokaryotes the flagellum is a
complex structure made of several proteins.
• Most of these proteins are anchored in the
cell wall and cytoplasmic membrane.
• The flagellum filament, which is made of a
single kind of protein, rotates at the expense
of the proton motive force, which drives the
flagellar motor.
Prokaryotes that move by gliding motility do
not employ rotating flagella, but instead creep
along a solid surface by any of several
possible mechanisms.
Motile bacteria can respond to chemical and
physical gradients in their environment.
• In the processes of chemotaxis and
phototaxis, random movement of a prokaryotic
cell can be biased either toward or away from
a stimulus by controlling the degree to which
runs or tumbles occur.
• The latter are controlled by the direction of
rotation of the flagellum, which in turn is
controlled by a network of sensory and
response proteins.
Microbial Metabolism
• Biocatalysis & Energy Generation
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Phosphorylation
Oxidation & Reduction
Fermentation & Respiration
Chemiosmosis: Proton Motive Force
ATPase Motor
Energy Yielding Metabolic Systems
Biosynthesis
∆G0' versus ∆G
standard conditions pH 7, 25°C
• The chemical reactions of the cell are
accompanied by changes in energy,
measured in kilojoules (kJ).
• A chemical reaction can occur with the
release of free energy (exergonic) or with
the consumption of free energy
(endergonic).
• 1 calorie = 4.186 Joules
Energy
G 0’f = free Energy of formation
for elements G 0’f = 0
ΔG 0’ = change in free Energy in reactions
ΔG 0’ of the reaction: A+B  C+D equals
ΔG 0’ [C+D] - ΔG 0’ [A+B]
products - reactants
if + , the reaction is ENDERGONIC
if - , the reaction is EXERGONIC
ΔG 0’ does not affect the rates of reaction
• The reactants in a chemical reaction must
first be activated before the reaction can take
place, and this requires a catalyst.
• Enzymes are catalytic proteins that speed up
the rate of biochemical reactions.
• Enzymes are highly specific in the reactions
they catalyze, and this specificity resides in
the three-dimensional structure of the
polypeptide(s) in the protein.
Enzyme Biocatalysis
• Specific substrate binding
• Substrate orientation o active sites
• Lowering the activation energy
Biological Energy Conservation
• The energy released in redox reactions is
conserved in the formation of certain
compounds that contain energy-rich
phosphate or sulfur bonds. The most
common of these compounds is ATP, the
prime energy carrier in the cell.
• Long-term storage of energy is linked to the
formation of polymers, which can be
consumed to yield ATP.
Microbial Metabolism
• Biocatalysis & Energy Generation
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Phosphorylation
Oxidation & Reduction
Fermentation & Respiration
Chemiosmosis: Proton Motive Force
ATPase Motor
Energy Yielding Metabolic Systems
Biosynthesis
REDOX potential
Oxidation–reduction reactions involve
the transfer of electrons from electron
donor to electron acceptor. The
tendency of a compound to accept or
release electrons is expressed
quantitatively by its reduction potential,
E0’.
• The transfer of electrons from donor to
acceptor in a cell typically involves one
or more electron carriers.
• Some electron carriers are membranebound, whereas others, such as
NAD+/NADH, are freely diffusible,
transferring electrons from one place to
another in the cell.
The energy released in redox reactions
is conserved in the formation of certain
compounds that contain energy-rich
phosphate or sulfur bonds.
• The most common of these compounds
is ATP, the prime energy carrier in the
cell.
• Long-term storage of energy is linked to
the formation of polymers, which can be
consumed to yield ATP.
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• Fermentation and respiration are the
two means by which chemoorganotrophs conserve energy from the
oxidation of organic compounds.
• During these catabolic reactions, ATP
synthesis occurs by way of either
substrate-level phosphorylation
(fermentation) or oxidative
phosphorylation (respiration).
• Glycolysis is a major pathway of
fermentation and is a widespread
means of anaerobic metabolism.
• The end result of glycolysis is the
release of a small amount of energy that
is conserved as ATP and the production
of fermentation products.
• For each glucose consumed in
glycolysis, 2 ATPs are produced.
Respiration involves the complete
oxidation of an organic compound with
much greater energy release than
during fermentation. The citric acid
cycle plays a major role in the
respiration of organic compounds.
• When electrons are transported through
an electron transport chain, protons are
extruded to the outside of the
membrane forming the proton motive
force.
• Key electron carriers include flavins,
quinones, the cytochrome bc1 complex,
and other cytochromes, depending on
the organism.
• The cell uses the proton motive force to
make ATP through the activity of
ATPase.
• Chemo –
Energy from chemical reactions
• Organo –
of organic compounds
• Hetero –
Carbon from organic sources
– trophic
feeding
• Electron acceptors other than O2 can
function as terminal electron acceptors
for energy generation. Because O2 is
absent under these conditions, the
process is called anaerobic respiration.
• Chemolithotrophs use inorganic
compounds as electron donors, while
phototrophs use light to form a proton
motive force.
• The proton motive force is involved in all
forms of respiration and photosynthesis.
Energy from:
Chemical reactions
or
Chemo-
Light
Photo-
of:
inorganic or organic
compounds
Litho-
Organo-
Source of carbon :
CO2
Auto-
or
(CH2O)n
Hetero-
Amino acids are formed from
carbon skeletons generated during
catabolism while nucleotides are
biosynthesized using carbon from
several sources.
Lipids
Fatty acids are synthesized two
carbons at a time and then attached to
glycerol to form lipids.