Chapter 4
Lecture Outline
Bacterial Culture, Growth,
and Development
Microbial Nutrition
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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

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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
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Obligate Intracellular Bacteria
SEM
Giemsa Stain
Rickettsia
Chicken fibroblast
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Microbial Nutrition: Source of Carbon

Heterotrophy
 Organic
compounds
 Generates and
releases CO2

Autotrophy
 CO2
from air
 CO2 fixation
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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
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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.
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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
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Microbiology: An Evolving Science
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Nutrient Up-Take
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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
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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
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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
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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
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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.
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Culturing Bacteria
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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
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Microbiology: An Evolving Science
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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:
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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
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Isolating Bacteria
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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

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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
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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
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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
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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)
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Bacterial Growth Curve
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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
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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
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Cell to Communication
Microbiology: An Evolving Science
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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
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Biofilm Formation in Pseudomonas
aeruginosa
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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)
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Cell Differentiation
Animation: Endospore Formation
Click box to launch animation
Microbiology: An Evolving Science
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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
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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
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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.
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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
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Concept Quiz
When food supplies dwindle, cells protect
themselves by forming
biofilms.
b. heterocysts.
c. spores.
a.
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