Microbiology_Ch_04_W2010 - Cal State LA
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Transcript Microbiology_Ch_04_W2010 - Cal State LA
Chapter 4
Lecture Outline
Bacterial Culture, Growth,
and Development
Microbial Nutrition
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition
All life requires:
Materials,
to
make cell
parts
Nutrients (C and others)
Energy,
to move
electrons
Electron flow, to drive all life processes
Drives ions into, out of cells
Used to create ATP
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition: Nutrients
Must be supplied from environment
Macronutrients
Major elements in cell macromolecules
Ions necessary for protein function
Trace elements necessary for enzyme function
Cobalt, manganese, nickel, zinc, etc.
Additional complex growth factors for fastidious
organisms
Mg2+, Ca2+, Fe2+, K+
Micronutrients
C, O, H, N, P, S
Amino acids, haemin, NAD, etc
Some bacteria need nitrogen as N2 gas from air
Some bacteria cannot be grown on artificial media
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Obligate Intracellular Bacteria
SEM
Giemsa Stain
Rickettsia
Chicken fibroblast
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition: Source of Carbon
Heterotrophy
Organic
compounds
Generates and
releases CO2
Autotrophy
CO2
from air
CO2 fixation
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition: Source of Energy
Phototrophs
Light
energy
Light absorption excites electrons to high energy
state
Perform photosynthesis
Chemotrophs
oxidation –reduction reactions
Transfer electrons from high energy
compounds to make products of lower energy
Chemical
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition: Electron Source
Lithotrophs
Inorganic
molecules are electron donors
Sulfur, iron, etc.
Organotrophs
Organic
molecules are electron donors
Glucose etc.
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbial Nutrition: Summary for
Prefixes for Term “-trophy”
Carbon source for biomass
Auto Hetero-
Energy source
Photo Chemo-
Electron source
Litho OrganoMicrobiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Nutrient Up-Take
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Nutrient Uptake: Diffusion
Passive diffusion
Some
gases pass freely through membranes
O2, CO2
Follows
gradient of material
Facilitated diffusion
Transporters
pass material
into/out of cell
Follows gradient of material
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Nutrient Uptake—Active Transport
Symport and Antiport
Gradient
of one molecule transports another
Electron transport creates Proton-Motive Force
PMF transports other molecules
Transports
Symport: Gradient
of
pumps
in
same direction
material against its gradient
Antiport: Gradient
of
pumps
in
opposite direction
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Nutrient Uptake—Active Transport
ABC Transporters
ATP
Binding Cassette
Use ATP energy to pass
material into cell
Transport material
against gradient
Used for uptake and
efflux
SBP only for up-take
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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siderophore
Siderophores and
Iron Up-Take System
Outer membrane protein
Periplasmic solutebinding protein
Siderophores have
high affinity for
soluble ferric ion
ABC transporter
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
15
Nutrient Uptake—Active Transport
Phosphotransferase System (PTS)
Uses ATP
energy to pass
material into cell
Modifies material as it
enters cell
Gradient is maintained,
pushing material into cell
glucose enters cell and is phosphorylated. As
a result, gradient of pushes more glucose
inside.
(glucose-6-phosphate) cannot pass out of cell.
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
16
Culturing Bacteria
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Culturing Bacteria
Culture media has all materials necessary
for growth
Varies
for different bacterial species
Electron source
Energy source
If not phototrophic
Carbon
source
If not autotrophic
Nitrogen
source
If not N2-fixer
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Growth Media
Complex media:
Exact
composition unknown
Examples: Beef bouillon, yeast extract
Enriched media
Contain
in addition blood components
Defined synthetic media:
Exact
composition known
Examples:
Microbiology: An Evolving Science
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Selective and Differential Media
Reveal differences in
metabolism
Selective
Suppresses
growth of unwanted
microbes
Differential
Includes
ingredients to detect
certain biochemical reactions
MacConkey
Selective and differential
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Isolating Bacteria
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Obtaining Pure Cultures
Dilution streaking
Streak
cells on plate
Agar inhibits spread of
microbes on plate
All cells in colony derive
from single cell
Genetically identical
Clone of that original cell
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Obtaining Pure Cultures
Dilution in liquid culture
Reduces
number of cells in each tube
Spread liquid on plate to see single colonies
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Determining the Concentration of
Bacteria
Counting
Petroff-Hauser chamber
Counts
cells directly
Gives accurate number
Can’t tell if cells are alive or dead
Use stain to distinguish living cells
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Determining the Concentration of
Bacteria
Spectrophotometer
Measures
optical density
“Shadow” of bacteria
Gives
rapid measurement
Can’t tell if cells are alive or dead
Solution must be at 107–1010 cells/ml
Drawing
of light
bulb
Photodetector
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Determining the Concentration of
Bacteria
Viable counts
Counts
only cells able to reproduce
Form colonies
Colony forming units (CFU/ml)
Assumes single cell suspension
Requires
time to form colonies (overnight)
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Bacterial Growth Curve
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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The Growth Cycle
Lag phase
Cells
synthesizing materials, not dividing
Log phase = exponential growth
1
2 4 8 16 …
10 doublings increases density by ~1000
log10(N) increases linearly
Stationary phase
Cells
no longer growing
Death phase
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
29
Stationary Phase
Total number of viable bacteria does not change
Changes in gene regulation
Quorum sensing induced
Biofilm formation
Species
Up regulation of virulence factors
specific
Spore formation
Cell differentiation
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Cell to Communication
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Biofilms
Cells secrete material to hold to a surface
Cells
Multiple species or a single species
Cells
acting together
signal to each other
Quorum sensing
Protects
against dispersion
Prevents antibiotics
from infiltrating
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Biofilm Formation in Pseudomonas
aeruginosa
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Endospore Formation
Cells respond to changing environment
Endospores
Protect against bad conditions
Disseminates cells
Forms
inside (“endo”) mother cell
Bacillus and Clostridium species
Exosporium
Spore coat (resistant,
calcium rich)
Cortex (peptidoglycan)
Dipicolinic acid
Small acid soluble
proteins (DNA
stabilizing)
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Cell Differentiation
Animation: Endospore Formation
Click box to launch animation
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Cell Differentiation
Cells respond to changing environment
Heterocysts
Different cells produce different
nutrients
Vegetative cells—energy
Heterocysts—fixed nitrogen
Myxospores
Form inside fruiting body
Multicellular structure
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Cell Differentiation
Cells respond to changing environment
Actinomycetes
form spores
Bacteria produce aerial hyphae
Specialized structures containing spores
When nutrients become limited
Protect genetic material
Disseminate cells
Spores
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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Concept Quiz
All bacterial cells need to be supplied with a
source of
electrons, energy, and nutrients.
b. carbon, nitrogen, and light.
c. carbon, fixed nitrogen, and water.
a.
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
38
Concept Quiz
The fastest way to measure cell density is
by using a
Petroff-Hauser counting chamber.
b. spectrophotometer.
c. petri plate.
a.
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
39
Concept Quiz
When food supplies dwindle, cells protect
themselves by forming
biofilms.
b. heterocysts.
c. spores.
a.
Microbiology: An Evolving Science
© 2009 W. W. Norton & Company, Inc.
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