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Chapter 6
Microbial Growth
1
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Growth
• increase in cellular constituents that may result
in:
– increase in cell number
• e.g., when microorganisms reproduce by
budding or binary fission
– increase in cell size
• e.g., coenocytic microorganisms have
nuclear divisions that are not
accompanied by cell divisions
• microbiologists usually study population
growth rather than growth of individual cells
2
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The Procaryotic Cell Cycle
• cell cycle is sequence of events from
formation of new cell through the next
cell division
– most bacteria divide by binary fission
• two pathways function during cycle
– DNA replication and partition
– cytokinesis
3
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Binary fission
4
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The Cell Cycle in E. coli
• E. coli requires ~40 minutes to replicate its DNA
and 20 minutes after termination of replication to
prepare for division
5
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Chromosome Replication and
Partitioning
• most procaryotic chromosomes are circular
• origin of replication – site at which replication
begins
• terminus – site at which replication is terminated
• replisome – group of proteins needed for DNA
synthesis; parent DNA spools through the
replisome as replication occurs
6
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Cell cycle of slow-growing E. coli
7
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Cytoskeletal Proteins
- Role in Cytokinesis
• process not well understood
• protein MreB
– similar to eucaryotic actin
– plays a role in determination of cell shape and
movement of chromosomes to opposite cell poles
• protein FtsZ,
– similar to eucaryotic tubulin
– plays a role in Z ring formation which is essential for
septation
8
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Cytoskeletal proteins involved in cytokinesis
in rod-shaped bacteria
MinCD, together with other
Min proteins, oscillates from
pole to pole
: prevents the formation of an
off-center Z ring
9
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Formation of the cell division apparatus in E. coli
10
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DNA Replication
in Rapidly Growing Cells
• cell cycle completed in 20 minutes
– 40 minutes for DNA replication
– 20 minutes for septum formation and
cytokinesis
• look at timing - how can this happen?
– second, third or fourth round of replication
can begin before first round of replication is
completed
11
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The Growth Curve
• Growth curve of microorganisms growing in a
batch culture (closed system)
– usually plotted as logarithm of cell number
versus time
– usually has four distinct phases
– batch culture: culture incubated in a closed
vessel with a single batch of medium
12
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Population growth ceases
Maximal rate of division
and population growth
No increase in cell number
13
Decline in
Population size
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Lag phase
• cell synthesizing new components
– e.g., to replenish spent materials
– e.g., to adapt to new medium or other
conditions
• varies in length
– in some cases can be very short or even
absent
14
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Exponential phase
• also called log phase
• rate of growth is constant
• population is most uniform in terms of
chemical and physical properties during
this phase
• cells exhibit balanced growth
– cellular constituents manufactured at
constant rates relative to each other
15
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Unbalanced growth
• rates of synthesis of cell components vary
relative to each other
• occurs under a variety of conditions
– change in nutrient levels
• shift-up (poor medium to rich medium)
• shift-down (rich medium to poor medium)
– change in environmental conditions
16
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Effect of nutrient concentration
on growth
Growth rate = number of generation/time
(k = n/t)
17
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Stationary phase
• total number of viable cells remains
constant
– may occur because metabolically active
cells stop reproducing
– may occur because reproductive rate is
balanced by death rate
18
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Possible reasons for entry into
stationary phase
•
•
•
•
19
nutrient limitation
limited oxygen availability
toxic waste accumulation
critical population density reached
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Starvation responses
• morphological changes
– e.g., endospore formation
• decrease in size, protoplast shrinkage, and
nucleoid condensation
• production of starvation proteins
- long-term survival
• increased virulence in pathogenic bacteria
20
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Death phase
• two alternative hypotheses on the
declining of viable cells during death
phase
1) cells are viable but nonculturable (VBNC)
: cells alive, but dormant (← genetic
response triggered in starving)
2) programmed cell death
– fraction of the population genetically
programmed to die (commit suicide)
21
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Loss of viability
22
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Prolonged decline in growth
• gradual decline instead of
exponential decline in
viability
• bacterial population
continually evolves
• process marked by successive
waves of genetically distinct
varients
• natural selection occurs
23
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The Mathematics of Growth
• generation (doubling) time (g)
– time required for the population to double in
size
– varies depending on species of
microorganism and environmental
conditions
– range is from 10 minutes for some bacteria
to several days for some eucaryotic
microorganisms
24
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25
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Exponential growth
26
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Generation time determination
27
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28
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Measurement of Microbial Growth
• can measure changes in number of
cells in a population
• can measure changes in mass of
population
29
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Measurement of Cell Numbers
• direct cell counts (total cell counts)
– counting chambers
– electronic counters
– on membrane filters
• viable cell counts
– plating methods
– membrane filtration methods
30
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Counting chambers
• easy, inexpensive, and
quick
• useful for counting
both eucaryotes and
procaryotes
• cannot distinguish
living from dead cells
Petroff-Hausser counting chamber
31
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Electronic counters
• Coulter counter and flow cytometer
• useful for large microorganisms and blood cells,
but not procaryotes
• microbial suspension forced through small orifice
• movement of microbe through orifice impacts
electric current that flows through orifice
• instances of disruption of current are counted
32
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Direct counts on membrane filters
• cells filtered through special membrane that
provides dark background for observing cells
(black polycarbonate membrane filter)
• cells are stained with fluorescent dyes
• useful for counting bacteria
• with certain dyes, can distinguish living from
dead cells
33
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Viable Counting Methods
• spread and pour plate techniques
– diluted sample of bacteria is spread over
solid agar surface or mixed with agar and
poured into Petri plate
– after incubation the numbers of organisms
are determined by counting the number of
colonies multiplied by the dilution factor
– results expressed as colony forming units
(CFU)
34
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Another viable count method
- membrane filtration
Especially useful for analyzing aquatic samples.
35
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Colonies on membrane filters
36
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Plating methods…
• simple and sensitive
• widely used for viable counts of
microorganisms in food, water, and
soil
• inaccurate results obtained if cells
clump together
37
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Measurement of cell mass
• dry weight
– time consuming and not very sensitive
• quantity of a particular cell constituent
– e.g., protein, DNA, ATP, or chlorophyll
– useful if amount of substance in each cell is
constant
• turbidometric measures (light scattering)
– quick, easy, and sensitive
38
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Absorbance (optical density)
more cells

more light
scattered

less light
detected
39
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The Continuous Culture of
Microorganisms
• growth in an open system
– continual provision of nutrients
– continual removal of wastes
• maintains cells in log phase at a constant
biomass concentration for extended
periods
• achieved using a continuous culture
system
40
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The chemostat
• rate of incoming
medium = rate of
removal of medium
from vessel
• an essential
nutrient is in
limiting quantities
41
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Dilution rate and microbial growth
• dilution rate
– rate at which medium flows
through vessel relative to
vessel size
• cell density remained
unchanged over a wide range
of dilution rates
• chemostat operates best at
low dilution rate
42
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The turbidostat
• regulates the flow rate of media through vessel
to maintain a predetermined turbidity or cell
density
• use a photocell to measure the absorbance or
turbidity
• dilution rate varies
• no limiting nutrient (all nutrients are present in
excess)
• turbidostat operates best at high dilution rates
43
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Importance of continuous culture
methods
• constant supply of cells in exponential phase
growing at a known rate
• study of microbial growth at very low nutrient
concentrations, close to those present in natural
environment
• study of interactions of microbes under conditions
resembling those in aquatic environments
• food and industrial microbiology
44
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The Influence of Environmental
Factors on Growth
• most organisms grow in fairly
moderate environmental conditions
• extremophiles
– grow under harsh conditions that
would kill most other organisms
45
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46
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Solutes and water activity
• water activity (aw)
– amount of water available to organisms
– reduced by interaction with solute
molecules (osmotic effect)
higher [solute]  lower aw
– reduced by adsorption to surfaces
(matric effect)
47
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Water activity (aw)
• water activity of a solution is 1/100 the
relative humidity of solution
• also equal to ratio of solution’s vapor
pressure (Psoln) to that of pure water
(Pwater)
• aw = Psoln/ Pwater
48
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49
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Osmotolerant organisms
• grow over wide ranges of water activity
• many use compatible solutes to increase
their internal osmotic concentration
– solutes that are compatible with metabolism
and growth
• some have proteins and membranes that
require high solute concentrations for
stability and activity
50
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Effects of NaCl on microbial growth
• halophiles
– grow optimally
at >0.2 M
• extreme halophiles
– require >2 M
51
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pH
52
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pH
• acidophiles
– growth optimum between pH 0 and pH 5.5
• neutrophiles
– growth optimum between pH 5.5 and pH 7
• alkalophiles
– growth optimum between pH 8.5 and pH 11.5
53
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pH
• most acidophiles and alkalophiles maintain an
internal pH near neutrality
– the plasma membrane is impermeable to protons
• some extremophiles synthesize proteins that
provide protection
– e.g., acid-shock proteins
• many microorganisms change pH of their
habitat by producing acidic or basic waste
products
– most media contain buffers to prevent growth
inhibition
54
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Temperature
• organisms exhibit
distinct cardinal
temperatures
– minimal
– maximal
– optimal
55
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56
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57
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Adaptations of thermophiles
• protein structure stabilized by a variety of
means
– e.g., more H bonds
– e.g., more proline
– e.g., chaperones
• histone-like proteins stabilize DNA
• membrane stabilized by variety of means
– e.g., more saturated, more branched and
higher molecular weight lipids
– e.g., ether linkages (archaeal membranes)
58
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Oxygen concentration
need
oxygen
59
prefer
oxygen
ignore
oxygen
oxygen is
toxic
< 2 – 10%
oxygen
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Basis of different oxygen
sensitivities
• oxygen easily reduced to toxic products
– superoxide radical
– hydrogen peroxide
– hydroxyl radical
• aerobes produce protective enzymes
– superoxide dismutase (SOD)
– catalase
60
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61
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62
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GasPak anaerobic system
63
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Pressure
• barotolerant
– adversely affected by increased pressure,
but not as severely as nontolerant
organisms
• barophilic organisms
– require or grow more rapidly in the
presence of increased pressure
64
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65
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Radiation
66
Electromagnetic spectrum
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Radiation damage
• ionizing radiation
– cause atoms to lose electrons (ionize)
– x-rays and gamma rays
– mutations  death
– disrupts chemical structure of many
molecules, including DNA
• damage may be repaired by DNA repair
mechanisms
67
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Radiation damage…
• ultraviolet (UV) radiation
– mutations  death
– causes formation of thymine dimers in
DNA
– DNA damage can be repaired by
several repair mechanisms
68
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Radiation damage…
• visible light
– at high intensities generates singlet
oxygen (1O2)
• powerful oxidizing agent
– carotenoid pigments
• protect many light-exposed
microorganisms from photooxidation
69
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Microbial Growth in
Natural Environments
• microbial environments are complex,
constantly changing, often contain low
nutrient concentrations (oligotrophic
environment) and may expose a
microorganism to overlapping gradients
of nutrients and environmental factors
70
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Growth limitation by
environmental factors
• Leibig’s law of the minimum
– total biomass of organism will be determined
by nutrient present at lowest concentration
• Shelford’s law of tolerance
– above or below certain environmental limits
(e.g., temperature), a microorganism will not
grow, regardless of the nutrient supply
71
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Responses to low nutrient levels
(oligotrophic environments)
• organisms become more competitive in
nutrient capture and use of available
resources
• morphological changes to increase
surface area and ability to absorb
nutrients
• mechanisms to sequester certain
nutrients
72
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Morphology and nutrient absorption (Caulobacter)
(a)
(b)
73
Stalks are relatively short when phosphorus is plentiful.
The stalks are extremely long under phosphorus-limited conditions.
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Counting viable but nonculturable
vegetative procaryotes
• stressed microorganisms can temporarily
lose ability to grow using normal
cultivation methods
• microscopic and isotopic methods for
counting viable but nonculturable cells
have been developed
74
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Counting and identifying microbes
in natural environments
– difficult to culture organisms from natural
environments
– previously stressed microbes are very
sensitive to secondary stresses and may not
grow on media normally used to cultivate
them
– Postgate microviability assay uses change in
morphology in a thin agar film under a
coverslip as indication of “life signs”
75
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Biofilms
• ubiquitous in nature
• complex, slime enclosed colonies attached
to surfaces
• when form on medical devices such as
implants often lead to illness
• can be formed on any conditioned surface
76
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(a)
(b)
77
Biofilm on the surface of a stromatolite. The film consists primarily of the cyanobacterium Calothrix.
Biofilm (white color) on the surface of artificial joint
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Biofilm formation
• microbes reversibly attach to conditioned
surface and release polysaccharides,
proteins, and DNA
• additional polymers are produced as
biofilm matures
• interactions occur among the attached
organisms
78
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Biofilm formation
79
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Biofilm microorganisms
• extracellular matrix and change in attached
organisms’ physiology protects them from
harmful agents such as UV light and
antibiotics
• sloughing off of organisms can result in
contamination of water phase above the
biofilm such as in a drinking water system
80
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Cell to cell communication
• acylhomoserine lactone (AHL) is an autoinducer
molecule produced by many gram-negative organisms
• AHL or other signal molecule diffuses across plasma
membrane
• AHL diffuses out of the cell at low cell density but
enters the cell when the cell population increases and
AHL accumulates outside the cell
• once inside the cell it induces expression of target genes
that regulate a variety of functions
81
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Quorum sensing
• influx of AHL is cell-density-dependent
• concentration present allows cells to access
population density
- process is called quorum sensing
82
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Representative cell-cell communication molecules
83