Chapter 5
Microbial Growth and Growth Control
I. Bacterial Cell Division
• 5.1 Binary Fission
• 5.2 Fts Proteins and Cell Division
• 5.3 MreB and Cell Morphology
• 5.4 Peptidoglycan Biosynthesis
5.1 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
5.2 Fts Proteins and Cell Division
• Fts (filamentous temperature-sensitive) proteins
(Figure
5.2)
• Essential for cell division in all prokaryotes
• Interact to form the divisome (cell division apparatus)
• FtsZ: forms ring around center of cell; related to tubulin
© 2015 Pearson Education, Inc.
• ZipA: anchor that connects FtsZ ring to cytoplasmic membrane
• FtsA: helps connect FtsZ ring to membrane and also recruits
other divisome proteins
•
Related to actin
5.2 Fts Proteins and Cell Division
• DNA replicates before the FtsZ ring forms (Figure 5.3)
• Location of FtsZ ring is facilitated by Min proteins
•
•
MinC, MinD, MinE
FtsK protein mediates separation of chromosomes to
daughter cells
5.3 MreB and Cell Morphology
• Prokaryotes contain a cell cytoskeleton that is dynamic
and multifaceted
•
MreB: major shape-determining factor in prokaryotes
• Forms simple cytoskeleton in Bacteria and probably
•
•
Archaea
Forms spiral-shaped bands around the inside of the cell,
underneath the cytoplasmic membrane (Figure 5.5a and b)
Not found in coccus-shaped bacteria
5.3 MreB and Cell Morphology
• MreB (cont'd)
•
Localizes synthesis of new peptidoglycan and other cell wall
components to specific locations along the cylinder of a rod© 2015 Pearson Education, Inc.
shaped cell during growth
5.3 MreB and Cell Morphology
• Crescentin: shape-determining protein produced by
vibrio-shaped cells of Caulobacter crescentus
• Crescentin protein organizes into filaments ~10 nm wide
that localize on the concave face of the curved cells (Figure
5.5c)
5.3 MreB and Cell Morphology
• Most archaeal genomes contain FtsZ and MreB-like
proteins; thus, cell morphology is similar to that seen in
Bacteria
5.4 Peptidoglycan Biosynthesis
• Production of new cell wall material is a major feature of
cell division
• In cocci, cell walls grow in opposite directions outward from
•
the FtsZ ring
In rod-shaped cells, growth occurs at several points along
length of the cell
5.4 Peptidoglycan Biosynthesis
• Preexisting peptidoglycan needs to be severed to allow
newly synthesized peptidoglycan to form (Figure 5.6)
© 2015 Pearson Education, Inc.
•
•
•
Beginning at the FtsZ ring, small openings in the wall are
created by autolysins
New cell wall material is added across the openings
Wall band: junction between new and old peptidoglycan
5.4 Peptidoglycan Biosynthesis
• Bactoprenol: carrier molecule that plays major role in
insertion of peptidoglycan precursors
• C55 alcohol (Figure 5.7)
• Bonds to N-acetylglucosamine/N-acetylmuramic
acid/pentapeptide peptidoglycan precursor
5.4 Peptidoglycan Biosynthesis
• Glycolases: enzymes that interact with bactoprenol
(Figure 5.8a)
• Insert cell wall precursors into growing points of cell wall
• Catalyze glycosidic bond formation
5.4 Peptidoglycan Biosynthesis
• Transpeptidation: final step in cell wall synthesis (Figure
5.8b)
• Forms the peptide cross-links between muramic acid
•
residues in adjacent glycan chains
Inhibited by the antibiotic penicillin
© 2015 Pearson Education, Inc.
II. Population Growth
• 5.5 Quantitative Aspects of Microbial Growth
• 5.6 The Growth Cycle
• 5.7 Continuous Culture
5.5 Quantitative Aspects of Microbial Growth
• Most bacteria have shorter generation times than
eukaryotic microbes
•
Generation time is dependent on growth medium and
incubation conditions
5.5 Quantitative Aspects of Microbial Growth
• 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 5.9)
5.5 Quantitative Aspects of Microbial 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
© 2015 Pearson Education, Inc.
N0 is the initial cell number
n is the number of generations during the
period of exponential growth
5.5 Quantitative Aspects of Microbial 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
5.5 Quantitative Aspects of Microbial Growth
• Specific growth rate (k) is calculated as
k = 0.301/g
•
Division rate (v) is calculated as
v = 1/g
5.6 The Growth Cycle
• 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
5.11):
• Lag phase
© 2015 Pearson Education, Inc.
•
•
•
Exponential phase
Stationary phase
Death phase
5.6 The Growth Cycle
• Lag phase
•
Interval between inoculation of a culture and beginning of
growth
•
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
5.6 The Growth Cycle
• Death phase
•
If incubation continues after cells reach stationary phase,
the cells will eventually die
5.7 Continuous Culture
• Continuous culture: an open-system microbial culture of
fixed volume
•
Chemostat: most common type of continuous culture
device (Figure 5.12)
© 2015 Pearson Education, Inc.
•
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
• Concentration of a limiting nutrient
5.7 Continuous Culture
• 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 5.13)
5.7 Continuous Culture
• Chemostat cultures are sensitive to the dilution rate and
limiting nutrient concentration (Figure 5.14)
• 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
III. Measuring Microbial Growth
• 5.8 Microscopic Counts
• 5.9 Viable Counts
© 2015 Pearson Education, Inc.
•
5.10 Spectrophotometry
5.8 Microscopic Counts
• Microbial cells are enumerated by microscopic
observations (Figure 5.15)
• Results can be unreliable
5.8 Microscopic Counts
• 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
5.8 Microscopic Counts
• A second method for enumerating cells in liquid samples
is use of a flow cytometer
• Uses laser beams, fluorescent dyes, and electronics
© 2015 Pearson Education, Inc.
5.9 Viable Counts
• Viable cell counts (plate counts): measurement of living,
reproducing population
• Two main ways to perform plate counts:
• Spread-plate method (Figure 5.16)
• Pour-plate method
•
To obtain the appropriate colony number, the sample to
be counted should always be diluted (Figure 5.17)
5.9 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
5.9 Viable Counts
• 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
© 2015 Pearson Education, Inc.
5.10 Spectrophotometry
• Turbidity measurements are indirect, rapid, and useful
methods of measuring microbial growth (Figure 5.18a)
• Most often turbidity is measured with a spectrophotometer,
and measurement is referred to as optical density (OD)
5.10 Spectrophotometry
• To relate a direct cell count to a turbidity value, a
standard curve must first be established (Figure 5.18c)
5.10 Spectrophotometry
• 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)
IV. Effect of Temperature on Microbial Growth
• 5.11 Temperature Classes of Microorganisms
• 5.12 Microbial Life in the Cold
• 5.13 Microbial Life at High Temperatures
© 2015 Pearson Education, Inc.
5.11 Temperature Classes of Microorganisms
• Temperature is a major environmental factor controlling
microbial growth
•
Cardinal temperatures: the minimum, optimum, and
maximum temperatures at which an organism grows
(Figure 5.19)
5.11 Temperature Classes of Microorganisms
• Microorganisms can be classified into groups by their
growth temperature optima (Figure 5.20)
• Psychrophile: low temperature
• Mesophile: midrange temperature
• Thermophile: high temperature
• Hyperthermophile: very high temperature
5.11 Temperature Classes of Microorganisms
• Mesophiles: organisms that have midrange temperature
optima; found in
• Warm-blooded animals
• Terrestrial and aquatic environments
• Temperate and tropical latitudes
5.12 Microbial Life in the Cold
• Extremophiles
•
Organisms that grow under very hot or very cold conditions
© 2015 Pearson Education, Inc.
•
Psychrophiles
• Organisms with cold temperature optima
• Inhabit permanently cold environments (Figure 5.21)
•
Psychrotolerant
• Organisms that can grow at 0ºC but have optima of 20ºC to
•
40ºC
More widely distributed in nature than psychrophiles
5.12 Microbial Life in the Cold
• Molecular adaptations that support psychrophily
•
Production of enzymes that function optimally in the cold;
features that may provide more flexibility
• More α-helices than β-sheets
• More polar and less hydrophobic amino acids
• Fewer weak bonds
• Decreased interactions between protein domains
5.12 Microbial Life in the Cold
• Molecular adaptations that support psychrophily (cont'd)
•
Transport processes function optimally at low temperatures
• Modified cytoplasmic membranes
• High unsaturated fatty acid content
5.13 Microbial Life at High Temperatures
• Above ~65ºC, only prokaryotic life forms exist
© 2015 Pearson Education, Inc.
•
Thermophiles: organisms with growth temperature
optima between 45ºC and 80ºC
•
Hyperthermophiles: organisms with optima greater than
80ºC
• Inhabit hot environments, including boiling hot springs and
seafloor hydrothermal vents, that can experience
temperatures in excess of 100ºC
5.13 Microbial Life at High Temperatures
• Hyperthermophiles in hot springs
•
•
Chemoorganotrophic and chemolithotrophic species are
present (Figure 5.23)
High prokaryotic diversity (both Archaea and Bacteria are
represented)
5.13 Microbial Life at High Temperatures
• Thermal gradients form along edges of hot environments
• Distribution of microbial species along the gradient is
dictated by organism's biology (Figure 5.24)
5.13 Microbial Life at High Temperatures
• Studies of thermal habitats have revealed:
•
•
Prokaryotes are able to grow at higher temperatures than
eukaryotes
Organisms with the highest temperature optima are
Archaea
© 2015 Pearson Education, Inc.
•
Nonphototrophic organisms can grow at higher
temperatures than phototrophic organisms
5.13 Microbial Life at High Temperatures
• 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 resists unfolding in the aqueous cytoplasm
• Production of solutes (e.g., di-inositol phosphate, diglycerol
phosphate) help stabilize proteins
5.13 Microbial Life at High Temperatures
• Molecular adaptations to thermophily (cont'd)
•
Modifications in cytoplasmic membranes to ensure heat
stability
• Bacteria have lipids rich in saturated fatty acids
• Archaea have lipid monolayer rather than bilayer
5.13 Microbial Life at High Temperatures
• Hyperthermophiles produce enzymes widely used in
industrial microbiology
• Example: Taq polymerase, used to automate the repetitive
steps in the polymerase chain reaction (PCR) technique
© 2015 Pearson Education, Inc.
V. Other Environmental Effects on Microbial
Growth
• 5.14 Effects of pH on Microbial Growth
• 5.15 Osmolarity and Microbial Growth
• 5.16 Oxygen and Microbial Growth
5.14 Effects of pH on Microbial Growth
• The pH of an environment greatly affects microbial
growth (Figure 5.25)
•
Some organisms have evolved to grow best at low or
high pH, but most organisms grow best between pH 6
and 8 (neutrophiles)
5.14 Effects of pH on Microbial Growth
• Acidophiles: organisms that grow best at low pH (<6)
•
•
•
Some are obligate acidophiles; membranes are destroyed
at neutral pH
Stability of cytoplasmic membrane is critical
Alkaliphiles: organisms that grow best at high pH (>9)
• Some have sodium motive force rather than proton motive
force
5.14 Effects of pH on Microbial Growth
© 2015 Pearson Education, Inc.
•
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 acidophiles and alkaliphiles, respectively
5.14 Effects of pH on Microbial Growth
• Microbial culture media typically contain buffers to
maintain constant pH
5.15 Osmolarity and Microbial Growth
• 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
5.15 Osmolarity and Microbial Growth
• 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
© 2015 Pearson Education, Inc.
5.15 Osmolarity and Microbial Growth
• Halophiles: organisms that grow best at reduced water
potential; have a specific requirement for NaCl (Figure
5.26)
•
Extreme halophiles: organisms that require high levels
(15–30%) 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
5.15 Osmolarity and Microbial Growth
• Osmophiles: organisms that live in environments high in
sugar as solute
•
Xerophiles: organisms able to grow in very dry
environments
5.15 Osmolarity and Microbial Growth
• Mechanisms for combating low water activity in
surrounding environment involve increasing the internal
solute concentration by
• Pumping inorganic ions from environment into cell
• Synthesizing or concentrating organic solutes
• Compatible solutes: compounds used by cell to counteract low
water activity in surrounding environment
© 2015 Pearson Education, Inc.
5.16 Oxygen and Microbial Growth
• 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
•
Microaerophiles: can use oxygen only when it is present
at levels reduced from that in air
Aerotolerant anaerobes: can tolerate oxygen and grow in
its presence even though they cannot use it
5.16 Oxygen and Microbial Growth
• Thioglycolate broth (Figure 5.27)
•
•
Complex medium that separates microbes based on oxygen
requirements
Reacts with oxygen, so oxygen can penetrate only the top
of the tube
5.16 Oxygen and Microbial Growth
• Special techniques are needed to grow aerobic and
anaerobic microorganisms (Figure 5.28)
•
Reducing agents: chemicals that may be added to
culture media to reduce oxygen (e.g., thioglycolate)
5.16 Oxygen and Microbial Growth
© 2015 Pearson Education, Inc.
•
Several toxic forms of oxygen can be formed in the cell
(Figure 5.29):
• Single oxygen
• Superoxide anion
• Hydrogen peroxide
• Hydroxyl radical
5.16 Oxygen and Microbial Growth
• Enzymes are present to neutralize most of these toxic
oxygen species (Figure 5.30)
• Catalase (Figure 5.31)
• Peroxidase
• Superoxide dismutase
• Superoxide reductase
V. Control of Microbial Growth
• 5.17 General Principles and Growth Control by Heat
• 5.18 Other Physical Control Methods: Radiation and
Filtration
•
5.19 Chemical Control of Microbial Growth
5.17 General Principles and Growth Control by
Heat
• Sterilization
© 2015 Pearson Education, Inc.
•
The killing or removal of all viable organisms within a
growth medium
•
Inhibition
• Effectively limiting microbial growth
•
Decontamination
• The treatment of an object to make it safe to handle
•
Disinfection
• Directly targets the removal of all pathogens, not
necessarily all microorganisms
5.17 General Principles and Growth Control by
Heat
• Heat sterilization is the most widely used method of
controlling microbial growth (Figure 5.32a)
• High temperatures denature macromolecules
• Amount of time required to reduce viability tenfold is
called the decimal reduction time (Figure 5.32b)
•
Some bacteria produce resistant cells called endospores
• Can survive heat that would rapidly kill vegetative
cells
5.17 General Principles and Growth Control by
Heat
• The autoclave is a sealed device that uses steam under
pressure (Figure 5.33)
• Allows temperature of water to get above 100ºC
• It's not the pressure, but the high temperature, that kills the
© 2015 Pearson Education, Inc.
microbes
•
Pasteurization is the process of using precisely
controlled heat to reduce the microbial load in heatsensitive liquids
• Does not kill all organisms, so it is different from sterilization
5.18 Other Physical Control Methods: Radiation
and Filtration
• Microwaves, UV, X-rays, gamma rays, and electrons can
reduce microbial growth
•
UV has sufficient energy to cause modifications and
breaks in DNA
• UV is useful for decontaminating surfaces (Figure 5.34)
• Cannot penetrate solid, opaque, or light-absorbing surfaces
5.18 Other Physical Control Methods: Radiation
and Filtration
• Ionizing radiation
•
•
•
Electromagnetic radiation that produces ions and other
reactive molecules
Generates electrons, hydroxyl radicals, and hydride
radicals
Some microorganisms are more resistant to radiation than
others
• Amount of energy required to reduce viability tenfold is
analogous to D value (Figure 5.35)
© 2015 Pearson Education, Inc.
5.18 Other Physical Control Methods: Radiation
and Filtration
• Sources of radiation include cathode ray tubes, X-rays,
and radioactive nuclides
•
Radiation is used for sterilization in the medical field and
food industry
• Radiation is approved by the WHO and is used in the USA
for decontaminating foods particularly susceptible to
microbial contamination
• Hamburger, chicken, spices may all be irradiated
5.18 Other Physical Control Methods: Radiation
and Filtration
• Filtration avoids the use of heat on sensitive liquids and
gases
• Pores of filter are too small for organisms to pass through
• Pores allow liquid or gas to pass through
•
Depth filters
• HEPA filters (Figure 5.36a)
•
Membrane filters
• Function more like a sieve (Figure 5.36b and c)
5.18 Other Physical Control Methods: Radiation
and Filtration
• Membrane filters (cont'd)
© 2015 Pearson Education, Inc.
•
•
Filtration can be accomplished by syringe, pump, or
vacuum (Figure 5.37)
A type of membrane filter is the nucleation track
(nucleopore) filter (Figure 5.38)
5.19 Chemical Control of Microbial Growth
• Antimicrobial agents can be classified as bacteriostatic,
bacteriocidal, and bacteriolytic (Figure 5.39)
5.19 Chemical Control of Microbial Growth
• Minimum inhibitory concentration (MIC) is the smallest
amount of an agent needed to inhibit growth of a
microorganism (Figure 5.40)
• Varies with the organism used, inoculum size,
temperature, pH, etc.
•
Disc diffusion assay uses solid media (Figure 5.41)
• Antimicrobial agent added to filter paper disc
• MIC is reached at some distance
•
Zone of inhibition
•
Area of no growth around disc
5.19 Chemical Control of Microbial Growth
• These antimicrobial agents can be divided into two
categories
• Products used to control microorganisms in commercial
and industrial applications
© 2015 Pearson Education, Inc.
•
•
Examples: chemicals in foods, air conditioning cooling
towers, textile and paper products, fuel tanks
Products designed to prevent growth of human pathogens
in inanimate environments and on external body surfaces
• Sterilants, disinfectants, sanitizers, and antiseptics
© 2015 Pearson Education, Inc.