Gim Lec Chapter 5

21.10.
2012
LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Chapter 5
Microbial Growth
Lectures by
John Zamora
Middle Tennessee State University
2012 Pearson Education, Inc.
I. Bacterial Cell Division

5.1 Cell Growth and Binary Fission
2012 Pearson Education, Inc. Marmara University Enve303 Env. Eng. Microbiology Prof. BARI ALLI
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5.1 Cell Growth and Binary Fission

Growth: increase in the number of cells
Binary fission: cell division following enlargement
of a cell to twice its minimum size (Figure 5.1)
Generation time: time required for microbial cells
to double in number
During cell division, each daughter cell receives a
chromosome and sufficient copies of all other cell
constituents to exist as an independent cell
Animation: Overview of Bacterial Growth
Animation: Binary Fission
Figure 5.1 Binary fission in a rod-shaped prokaryote
Cell
elongation
One generation
Septum
Septum formation
Completion
of septum;
formation of
walls; cell
separation
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II. Population Growth

5.5 The Concept of Exponential Growth
5.6 The Mathematics of Exponential Growth
5.7 The Microbial Growth Cycle
5.8 Continuous Culture: The Chemostat

Most bacteria have shorter generation times
than eukaryotic microbes
Generation time is dependent on growth
medium and incubation conditions
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Exponential growth: growth of a microbial
population in which cell numbers double
within a specific time interval
During exponential growth, the increase in
cell number is initially slow but increases at a
faster rate (Figure 5.8)
Figure 5.8 The rate of growth of a microbial culture
1000
(logarithmic scale)
(arithmetic scale)
103
Number of cells
Logarithmic
Number of cells
plot
Arithmetic 102
500 plot
10
100
0 1 2 3 4 5 1
Time (h)
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5.6 The Mathematics of Exponential

Growth
A relationship exists between the initial number of
cells present in a culture and the number present
after a period of exponential growth:
N = N02n
N is the final cell number
N0 is the initial cell number
n is the number of generations during the
period of exponential growth

Growth
Generation time (g) of the exponentially
growing population is
g = t/n
t is the duration of exponential growth
n is the number of generations during
the period of exponential growth
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Growth
Specific growth rate (k) is log(2)
k Slope 0.15
2
calculated as
k = 0.301/g
Division rate (v) is
calculated as
v = 1/g
Figure 5.9 Calculating microbial growth parameters.
Method of estimating the generation times (g) of
exponentially growing populations with generation
times of 2 h from data plotted on semilogarithmic
graphs. The slope of line is equal to 0.30/g, and n is
the number of generations in the time t.

Batch culture: a closed-system microbial culture
of fixed volume
Typical growth curve for population of cells grown
in a closed system is characterized by four
phases (Figure 5.10):
Lag phase
Exponential phase
Stationary phase
Death phase
Animation: Bacterial Growth Curve
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Figure 5.10 Typical growth curve for a bacterial population
Growth phases
Lag Exponential Stationary Death
1.0
Optical density (OD)

10
0.75
organisms/ml
Log10 viable
9 Turbidity
(optical density) 0.50
Viable count
8
0.25
6
0.1
Time
A viable count measures the cells in the culture that are capable of
reproducing. Optical density (turbidity), a quantitative measure of light
scattering by a liquid culture, increases with the increase in cell number.

Lag phase
Interval between when a culture is inoculated
and when growth begins
Exponential phase
Cells in this phase are typically in the
healthiest state
Stationary phase
Growth rate of population is zero
Either an essential nutrient is used up or
waste product of the organism accumulates
in the medium
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Death Phase
If incubation continues after cells reach
stationary phase, the cells will eventually die

Continuous culture: an open-system microbial
culture of fixed volume
Chemostat: most common type of continuous
culture device (Figure 5.11)
Both growth rate and population density of culture
can be controlled independently and
simultaneously
Dilution rate: rate at which fresh medium is pumped
in and spent medium is pumped out (mean cell
residence time or hydraulic retention time HRT)
Concentration of a limiting nutrient
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Figure 5.11 Schematic for a continuous culture device (chemostat)
Fresh medium Flow-rate

from reservoir regulator
Sterile air or
other gas Gaseous
headspace
Culture
vessel
Culture
Overflow
Effluent containing
microbial cells
The population density is controlled by the concentration of limiting

nutrient in the reservoir, and the growth rate is controlled by the flow rate.
Both parameters can be set by the experimenter.

In a chemostat
The growth rate is controlled by dilution rate
The growth yield (cell number/ml) is controlled
by the concentration of the limiting nutrient
In a batch culture, growth conditions are
constantly changing; it is impossible to
independently control both growth
parameters (Figure 5.12)
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Figure 5.12 The effect of nutrients on growth
Rate and Only yield affected

yield affected
)
)
Growth rate (
Growth yield (
0 0.1 0.2 0.3 0.4 0.5
Nutrient concentration (mg/ml)
Relationship between nutrient concentration, growth rate (green curve),
and growth yield (red curve) in a batch culture (closed system). Only at
low nutrient concentrations are both growth rate and growth yield affected.

Chemostat cultures are sensitive to the dilution
rate and limiting nutrient concentration
(Figure 5.13)
At too high a dilution rate, the organism is
washed out
At too low a dilution rate, the cells may die from
starvation
Increasing concentration of a limiting nutrient
results in greater biomass but same growth
rate
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Figure 5.13 Steady-state relationships in the chemostat
Steady state
Steady -state bacterial concentration (g/l)

5 Bacterial concentration
4 6
Doubling time (h)

3
4
2
2
1
0 0
0 0.25 0.5 0.75 1.0
Dilution rate (h 1) Washout
At high dilution rates, growth cannot balance dilution, and the population
washes out. Although the population density remains constant during
steady state, the growth rate (doubling time) can vary over a wide range.
III. Measuring Microbial Growth

5.9 Microscopic Counts
5.10 Viable Counts
5.11 Turbidimetric Methods
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Microbial cells are enumerated by microscopic
observations (Figure 5.14)
Results can be unreliable
Figure 5.14 Direct microscopic counting procedure using the PetroffHausser counting chamber
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Limitations of microscopic counts
Cannot distinguish between live and dead cells
without special stains
Small cells can be overlooked
Precision is difficult to achieve
Phase-contrast microscope required if a stain is
not used
Cell suspensions of low density (<106 cells/ml)
hard to count
Motile cells need to immobilized
Debris in sample can be mistaken for cells

A second method for enumerating cells in
liquid samples is with a flow cytometer
Uses laser beams, fluorescent dyes, and
electronics
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5.10 Viable Counts

Viable cell counts (plate counts):
measurement of living, reproducing
population
Two main ways to perform plate counts:
Spread-plate method (Figure 5.15)
Pour-plate method
To obtain the appropriate colony number, the
sample to be counted should always be
diluted (Figure 5.16)
Figure 5.15 Two methods for the viable count
Spread-plate method
Surface
colonies
Incubation
Sample is pipetted onto Sample is spread evenly Typical spread-plate Colonies of

surface of agar plate over surface of agar results Escherichia coli
(0.1 ml or less) using sterile glass formed from cells
spreader plated by the spread-
plate method (top) or
Pour-plate method the pour-plate
method (bottom)
Surface
colonies
Solidification
and incubation
Subsurface
colonies
Sample is pipetted into Sterile medium is Typical pour-plate Colonies form within
sterile plate added and mixed results the agar as well as
well with inoculum on the agar surface
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Figure 5.16 Procedure for viable counting using serial dilutions of the sample and the pour-plate method
The sterile liquid used for

making dilutions can simply
be water, but a solution of
Sample to
1 ml be counted mineral salts or actual
growth medium may yield a
1 ml 1 ml 1 ml 1 ml 1 ml higher recovery.
9-ml
broth
Total 1/10 1/100 1/103 1/104 1/105 1/106

dilution (101) (102) (103) (104) (105) (106)
Plate 1-ml samples
159 17 2 0
Too many colonies colonies colonies colonies colonies
to count
159 103 1.59 105
Plate Dilution Cells (colony-forming units)
count factor per milliliter of original sample
5.10 Viable Counts

Plate counts can be highly unreliable when
used to assess total cell numbers of natural
samples (e.g., soil and water)
Selective culture media and growth conditions
target only particular species
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5.10 Viable Cell Counting

The Great Plate Anomaly: direct microscopic
counts of natural samples reveal far more
organisms than those recoverable on plates
Why is this?
Microscopic methods count dead cells
whereas viable methods do not
Different organisms may have vastly different
requirements for growth

Turbidity measurements are an indirect, rapid,
and useful method of measuring microbial
growth (Figure 5.17a)
Most often measured with a spectrophotometer
and measurement referred to as optical density
(O.D.)
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Figure 5.17a Turbidity measurements of microbial growth
Measurements of turbidity
Light
are made in a
spectrophotometer.
The photocell measures
Prism
incident light unscattered by
cells in suspension and
Incident light, I0
gives readings in optical
Filter density units.
Sample containing
cells ( )
Unscattered light, I
Photocell (measures
unscattered light, I )
Spectrophotometer
Optical density (OD)

I0
Log
I

To relate a direct cell count to a turbidity
value, a standard curve must first be
established (Figure 5.17c)
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Figure 5.17c Turbidity measurements of microbial growth
Theoretical
0.8
Optical density 0.7 The one-to one
Actual
0.6 correspondence between
0.5 these relationships breaks
0.4
down at high turbidities
0.3
0.2
0.1
Cell numbers or mass

(dry weight)
Relationship between cell

number or dry weight and
turbidity readings.

Turbidity measurements
Quick and easy to perform
Typically do not require destruction or
significant disturbance of sample
Sometimes problematic (e.g., microbes that
form clumps or biofilms in liquid medium)
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IV. Temperature and Microbial

Growth
5.12 Effect of Temperature on Growth
5.13 Microbial Life in the Cold
5.14 Microbial Life at High Temperatures

Temperature is a major environmental factor
controlling microbial growth
Cardinal temperatures: the minimum,
optimum, and maximum temperatures at
which an organism grows (Figure 5.18)
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Figure 5.18 The cardinal temperatures: minimum, optimum, and maximum
Enzymatic reactions occurring

at maximal possible rate
Optimum
Growth rate
Enzymatic reactions occurring

at increasingly rapid rates
Actual
values
may vary
Minimum Maximum greatly for
different
organisms
Temperature
Membrane gelling; transport Protein denaturation; collapse

processes so slow that growth of the cytoplasmic membrane;
cannot occur thermal lysis

Microorganisms can be classified into groups by
their growth temperature optima (Figure 5.19)
Psychrophile: low temperature
Mesophile: midrange temperature
Thermophile: high temperature
Hyperthermophile: very high temperature
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Figure 5.19 Temperature and growth response in different temperature classes of microorganisms
Thermophile
Example:
Geobacillus Hyperthermophile Hyperthermophile
stearothermophilus Example: Example:
Mesophile Thermococcus celer Pyrolobus fumarii
Example: 60
Growth rate
Escherichia coli 106

88
Psychrophile 39
Example:
Polaromonas vacuolata
0 10 20 30 40 50 60 70 80 90 100 110 120

Temperature (oC)

Mesophiles: organisms that have midrange
temperature optima; found in
Warm-blooded animals
Terrestrial and aquatic environments
Temperate and tropical latitudes
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Extremophiles
Organisms that grow under very hot or very cold
conditions
Psychrophiles
Organisms with cold temperature optima
Inhabit permanently cold environments
(Figure 5.20)
Psychrotolerant
Organisms that can grow at 0C but have optima of
20C to 40C
More widely distributed in nature than
psychrophiles

Molecular Adaptations to Psychrophily
Production of enzymes that function optimally
in the cold; features that may provide more
flexibility
More -helices than -sheets
More polar and less hydrophobic amino acids
Fewer weak bonds
Decreased interactions between protein
domains
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Molecular Adaptations to Psychrophily (contd)
Transport processes function optimally at low
temperatures
Modified cytoplasmic membranes
High unsaturated fatty acid content

Above ~65 oC, only prokaryotic life forms exist
Thermophiles: organisms with growth temperature
optima between 45 oC and 80 oC
Hyperthermophiles: organisms with optima greater
than 80 oC
Inhabit hot environments including boiling hot
springs and seafloor hydrothermal vents that can
have temperatures in excess of 100 oC
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Hyperthermophiles in Hot Springs
Chemoorganotrophic and chemolithotrophic
species present (Figure 5.22)
High prokaryotic diversity (both Archaea and
Bacteria represented)
Figure 5.22 Growth of hyperthermophiles in boiling water
(a) Boulder Spring, a

small boiling spring in
Yellowstone National
Park. This spring is
superheated, having a
temperature 1-2 oC above
the boiling point. The
mineral deposits around
the spring consist mainly
of silica and sulfur.
(b) Photomicrograph of a
microcolony of
prokaryotes that
developed on a
microscope slide
immersed in such a
boiling spring.
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Studies of thermal habitats have revealed
Prokaryotes are able to grow at higher
temperatures than eukaryotes
Organisms with the highest temperature
optima are Archaea
Nonphototrophic organisms can grow at
higher temperatures than phototrophic
organisms

Molecular Adaptations to Thermophily
Enzyme and proteins function optimally at high
temperatures; features that provide thermal
stability
Critical amino acid substitutions in a few locations
provide more heat-tolerant folds
An increased number of ionic bonds between basic
and acidic amino acids resist unfolding in the
aqueous cytoplasm
Production of solutes (e.g., di-inositol phophate,
diglycerol phosphate) help stabilize proteins
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Molecular Adaptations to Thermophily (contd)
Modifications in cytoplasmic membranes to
ensure heat stability
Bacteria have lipids rich in saturated fatty acids
Archaea have lipid monolayer rather than bilayer
V. Other Environmental Factors

Affecting Growth
5.15 Acidity and Alkalinity
5.16 Osmotic Effects on Microbial Growth
5.17 Oxygen and Microorganisms
5.18 Toxic Forms of Oxygen
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The pH of an environment greatly affects
microbial growth (Figure 5.24)
Some organisms have evolved to grow best
at low or high pH, but most organisms grow
best between pH 6 and 8 (neutrophiles)
Figure 5.24 The pH scale
pH Example Moles per liter of:

H OH
1 1014
Volcanic soils, waters
Gastric fluids
101 1013
Acidophiles
Lemon juice
Acid mine drainage 102 1012
Vinegar
Increasing Rhubarb 103 1011
acidity Peaches
Acid soil 104 1010
Tomatoes Although some
American cheese 105 109
Cabbage microorganisms
Peas 106 108 can live at very low
Corn, salmon, shrimp
Neutrality Pure water 107 107 or very high pH,
Seawater 108 106 the cells internal
105
pH remains near
Very alkaline 109
natural soil neutrality.
Alkaliphiles
Alkaline lakes 1010 104

Increasing Soap solutions
alkalinity Household ammonia 1011 103
Extremely alkaline
soda lakes 1012 102
Lime (saturated solution)
1013 101
1014 1
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Acidophiles: organisms that grow best at low
pH (<6)
Some are obligate acidophiles; membranes
destroyed at neutral pH
Stability of cytoplasmic membrane critical
Alkaliphiles: organisms that grow best at high
pH (>9)
Some have sodium motive force rather than
proton motive force

The internal pH of a cell must stay relatively
close to neutral even though the external pH
is highly acidic or basic
Internal pH has been found to be as low as
4.6 and as high as 9.5 in extreme acido- and
alkaliphiles, respectively
Microbial culture media typically contain
buffers to maintain constant pH
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Water activity (aw): water availability;
expressed in physical terms
Defined as ratio of vapor pressure of air in
equilibrium with a substance or solution to the
vapor pressure of pure water

Typically, the cytoplasm has a higher solute
concentration than the surrounding
environment, thus the tendency is for water to
move into the cell (positive water balance)
When a cell is in an environment with a higher
external solute concentration, water will flow
out unless the cell has a mechanism to
prevent this
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Halophiles: organisms that grow best at reduced
water potential; have a specific requirement for
NaCl (Figure 5.25)
Extreme halophiles: organisms that require high

levels (1530%) of NaCl for growth
Halotolerant: organisms that can tolerate some

reduction in water activity of environment but
generally grow best in the absence of the added
solute
Figure 5.25 Effect of sodium chloride (NaCl) concentration on growth of microorganisms of different salt
tolerances or requirements.
Halotolerant Halophile Extreme

Example: Example: halophile
Staphylococcus Aliivibrio fischeri Example:
aureus Halobacterium
salinarum
Growth rate
Nonhalophile
Example:
Escherichia coli
0 5 10 15 20
NaCl (%)
Optimum NaCl concentration for extreme halophiles, it is between 15-30%
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Osmophiles: organisms that live in
environments high in sugar as solute
Xerophiles: organisms able to grow in very
dry environments

Mechanisms for combating low water activity in
surrounding environment involve increasing the
internal solute concentration by
Pumping inorganic ions from environment
into cell
Synthesis or concentration of organic solutes
compatible solutes: compounds used by cell to
counteract low water activity in surrounding
environment
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Aerobes: require oxygen to live
Anaerobes: do not require oxygen and may even
be killed by exposure
Facultative organisms: can live with or without
oxygen
Aerotolerant anaerobes: can tolerate oxygen and
grow in its presence even though they cannot
use it
Microaerophiles: can use oxygen only when it is
present at levels reduced from that in air

Thioglycolate broth (Figure 5.26)
Complex medium that separates microbes
based on oxygen requirements
Reacts with oxygen so oxygen can only
penetrate the top of the tube
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Figure 5.26 Growth versus oxygen (O2) concentration
The redox dye, resazurin, which is pink when oxidized and colorless when reduced, has
been added as a redox indicator.
(a) O2 penetrates only a short
distance into the tube, so
obligate aerobes grow only
close to the surface.
(b) Anaerobes, being sensitive to
O2, grow only away from the
surface.
(c) Facultative aerobes are able to
Oxic zone grow in either the presence or
Aerotolerant anaerobes
the absence of O2 and thus
Facultative aerobes
grow throughout the tube.

However, growth is better near
Microaerophiles
the surface because these

Anoxic zone organisms can respire.
Anaerobes
(d) Microaerophiles grow away

Aerobes
from the most oxic zone.

(e) Aerotolerant anaerobes grow
throughout the tube. Growth is
not better near the surface
because these organisms can
only ferment.

Special techniques are needed to grow
aerobic and anaerobic microorganisms
(Figure 5.27)
Reducing agents: chemicals that may be
added to culture media to reduce oxygen
(e.g., thioglycolate)
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Figure 5.27 Incubation under anoxic conditions
Anoxic jar Anoxic glove bag
A chemical reaction in the envelope Anoxic glove bag for manipulating and incubating cultures under anoxic
in the jar generates H2 + CO2. The H2 conditions. The airlock on the right, which can be evacuated and filled
reacts with O2 in the jar on the with O2-free gas, serves as a port for adding and removing materials to
surface of a palladium catalyst to and from the glove bag.
yield H2O; the final atmosphere
contains N2, H2, and CO2.

Several toxic forms of oxygen can be formed
in the cell (Figure 5.28):
Single oxygen
Superoxide anion
Hydrogen peroxide
Hydroxyl radical
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Figure 5.28 Four-electron reduction of O2 to H2O by stepwise addition of electrons
Reactants Products
(superoxide)
(hydrogen peroxide)
(hydroxyl radical)
(water)
Outcome:
All intermediates formed are reactive and toxic to cells, except for water

Enzymes are present to neutralize most of
these toxic oxygen species
Catalase
Peroxidase
Superoxide dismutase
Superoxide reductase
35

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21.10.

2012 Pearson Education, Inc.

I. Bacterial Cell Division

5.1 Cell Growth and Binary Fission

Animation: Binary Fission

Figure 5.1 Binary fission in a rod-shaped prokaryote

II. Population Growth

5.5 The Concept of Exponential Growth

5.5 The Concept of Exponential Growth

Figure 5.8 The rate of growth of a microbial culture

5.6 The Mathematics of Exponential

5.6 The Mathematics of Exponential

5.6 The Mathematics of Exponential

5.7 The Microbial Growth Cycle

Animation: Bacterial Growth Curve

Figure 5.10 Typical growth curve for a bacterial population

Optical density (OD)

5.7 The Microbial Growth Cycle

5.7 The Microbial Growth Cycle

5.8 Continuous Culture: The Chemostat

Figure 5.11 Schematic for a continuous culture device (chemostat)

Fresh medium Flow-rate

The population density is controlled by the concentration of limiting

5.8 Continuous Culture: The Chemostat

Figure 5.12 The effect of nutrients on growth

Rate and Only yield affected

5.8 Continuous Culture: The Chemostat

Figure 5.13 Steady-state relationships in the chemostat

Steady -state bacterial concentration (g/l)

Doubling time (h)

III. Measuring Microbial Growth

5.9 Microscopic Counts

5.9 Microscopic Counts

5.9 Microscopic Counts

5.10 Viable Counts

Figure 5.15 Two methods for the viable count

Sample is pipetted onto Sample is spread evenly Typical spread-plate Colonies of

The sterile liquid used for

Total 1/10 1/100 1/103 1/104 1/105 1/106

5.10 Viable Counts

5.10 Viable Cell Counting

5.11 Turbidimetric Methods

Figure 5.17a Turbidity measurements of microbial growth

Optical density (OD)

5.11 Turbidimetric Methods

Figure 5.17c Turbidity measurements of microbial growth

Cell numbers or mass

Relationship between cell

5.11 Turbidimetric Methods

IV. Temperature and Microbial

5.12 Effect of Temperature on Growth

Figure 5.18 The cardinal temperatures: minimum, optimum, and maximum

Enzymatic reactions occurring

Enzymatic reactions occurring

Membrane gelling; transport Protein denaturation; collapse

5.12 Effect of Temperature on Growth

Escherichia coli 106

0 10 20 30 40 50 60 70 80 90 100 110 120

5.12 Effect of Temperature on Growth

5.13 Microbial Life in the Cold

5.13 Microbial Life in the Cold

5.13 Microbial Life in the Cold

5.14 Microbial Life at High Temperatures

5.14 Microbial Life at High Temperatures