4ch06 Lecture

Brock Biology of Microorganisms
Twelfth Edition
Madigan / Martinko Dunlap / Clark
Chapter 6
Microbial Growth
Lectures by Buchan & LeCleir
Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings
I. Bacterial Cell Division

6.1 Cell Growth and Binary Fission
6.2 Fts Proteins and Cell Division

6.3 MreB and Determinants of Cell Morphology
6.4 Peptidoglycan Synthesis and Cell Division
6.1 Cell Growth and Binary Fission

Growth: increase in the number of cells Binary fission: cell elongation following enlargement of a cell to twice its minimum size Generation time: time required for a population of microbial cells to double 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
Binary Fission in a Rod-Shaped Prokaryote
Figure 6.1
6.3 Skip Section
Peptidoglycan Synthesis
Figure 6.7a
6.4 Peptidoglycan Synthesis and Cell Division
Transpeptidation: final step in cell wall synthesis

Forms the peptide cross-links between muramic acid
residues in adjacent glycan chains Inhibited by the penicillin & cephalosporin antibiotic classes [aka -lactams]
Peptidoglycan Synthesis
Figure 6.7b
II. Growth of Bacterial Populations

6.5 Growth Terminology and the Concept of Exponential
Growth
6.6 The Mathematics of Exponential Growth 6.7 The Microbial Growth Cycle 6.8 Continuous Culture: The Chemostat
6.5 Growth Terminology and the Concept of Exponential Growth
Most bacteria have shorter generation times than
eukaryotic microbes
Generation time is dependent on growth medium and incubation conditions
6.5 Growth Terminology and the Concept of Exponential Growth
Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval Generation time: the time it take for the cell population to double
The Rate of Growth of a Microbial Culture
Figure 6.8
Calculating Microbial Growth Parameters
Figure 6.9
6.6 The Mathematics of Exponential Growth

Increase in cell number in an exponentially growing bacterial culture is a geometric progression of the number 2 Relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth:
N = No2n
where N is the final cell number, No is the initial cell number, and n is the number of generations during the period of exponential growth
6.6 The Mathematics of Exponential Growth

Generation time (g) of the exponentially growing population is
g = t/n where t is the duration of exponential growth
and n is the number of generations during the

period of exponential growth
6.7 The Microbial Growth Cycle

Typical growth curve for population of cells grown in a closed system is characterized by four phases
Lag phase Exponential phase Stationary phase
Death phase
Animation: Bacterial Growth Curve
Typical Growth Curve for a Bacterial Population
Figure 6.10
Lag phase
Interval of time 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
Death phase
Rate of cell death is greater than growth rate producing a
decrease in cell numbers
III. Measuring Microbial Growth

6.9 Measurements of Total Cell Numbers: Microscopic
Counts
6.10 Viable Cell Counting 6.11 Measurements of Microbial Mass: Turbidimetric Methods
6.9 Measurements of Total Cell Numbers: Microscopic Counts

Microbial cells can be enumerated by microscopic
observations; simple but results can be unreliable Limitations of microscopic counts
Cannot distinguish between live and dead cells without special stains
Small cells difficult to see and can be overlooked

Precision is difficult to achieve A phase-contrast microscope is required if a stain is not used
Limitations of microscopic counts (contd)

Cell suspensions of low density (< 106 cells/ml) hard to
count Motile cells need to immobilized Debris in sample can be mistaken for cells
Direct Microscopic Counting Procedure
Figure 6.14

A second method for enumerating cells in liquid
samples is with a flow cytometer
6.10 Viable Cell Counting

Viable cell counts (plate counts): measurement of living,
reproducing population
Two main ways to perform plate counts
Spread-plate method Pour-plate method
To obtain the appropriate colony number, the sample to be counted should always be diluted
Spread-Plate Method for the Viable Count
Figure 6.15
Pour-Plate Method for the Viable Count
Figure 6.15
Procedure for Viable Counting Using Serial Dilutions
Figure 6.16
Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (e.g., soil and water)
The Great Plate Anomaly: direct microscopic counts of

natural samples typically reveal far more organisms
than are recoverable on plates of any given culture

medium
Why is this?
Microscopic methods count dead cells whereas viable
methods do not Different organisms in even a very small sample may have vastly different requirements for resources and conditions in laboratory culture
6.11 Measurements of Microbial Mass: Turbidimetric Methods
Turbidity measurements are an indirect but very
rapid and useful method of measuring microbial

growth
Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.)
Turbidity Measurements of Microbial Growth
Figure 6.17a
6.11 Measurements of Microbial Mass: Turbidimetric Methods
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)
Typical Growth Curve for a Bacterial Population
Figure 6.10
IV. Temperature and Microbial Growth

6.12 Effects of Temperature on Microbial Growth
6.13 Microbial Growth at Cold Temperatures

6.14 Microbial Growth 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
The Cardinal Temperatures
Figure 6.18

Microorganisms can be classified into groups by
their growth temperature optima

Psychrophile: low temperature Mesophile: midrange temperature Thermophile: high temperature Hyperthermophile: very high temperature
Temperature and Growth Relations in Different Classes
Figure 6.19

Mesophiles: organisms that have midrange temperature
optima; found in
Warm-blooded animals Terrestrial and aquatic environments Temperate and tropical latitudes

Extremophiles
Organisms that have evolved to grow optimally under very
hot or very cold conditions
Psychrophiles
Organisms with cold temperature optima; the most extreme representatives inhabit permanently cold environments
Psychrotolerant
Organisms that can grow at 0C but have optima of 20C to 40C; more widely distributed in nature than psychrophiles
Antarctic Microbial Habitats and Microorganisms
Figure 6.20a and b

Molecular Adaptations to Psychrophily
Transport processes function optimally at low
temperatures due to modifications of cytoplasmic membranes
High unsaturated fatty acid content

Above ~65C only prokaryotic life forms exist
Thermophiles: organisms with growth temperature optima between 45C and 80C Hyperthermophiles: organisms with optima >80C Inhabit hot environments including boiling hot springs and seafloor hydrothermal vents that can have temperatures in excess of 100C
Upper Temperature Limits for Growth of Living Organisms
Upper Temperature Limits for Growth of Living Organisms

Hyperthermophiles in Hot Springs
Chemoorganotrophic and chemolithotrophic species
present High prokaryotic diversity (both Archaea and Bacteria represented)
Growth of Hyperthermophiles in Boiling Water
Figure 6.22

Thermal gradients form along edges of hot
environments
Distribution of microbial species along the gradient is dictated by organisms biology
Growth of Thermophilic Cyanobacteria in a Hot Spring
Figure 6.23
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
6.14 Microbial Growth at High Temperature

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, diglyercol phosphate) help stabilize proteins

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

Hyperthermophiles and produce enzymes widely used
in industrial microbiology
E.g., Taq polymerase; used to automate the repetitive steps in the polymerase chain reaction (PCR) technique
V. Other Environmental Factors Affecting Growth

6.15 Microbial Growth at Low or High pH
6.16 Osmotic Effects on Microbial Growth

6.17 Oxygen and Microbial Growth
6.18 Toxic Forms of Oxygen

The pH of an environment greatly affects microbial
growth
Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)
The pH Scale
Figure 6.24

Acidophiles: organisms that grow best at low pH (< 6)
Some 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 high as 9.5 in extreme acido- and alkaliphiles, respectively
Microbial culture media typically contain buffers to
maintain constant pH
Water activity
The ratio of vapor pressure of air in equilibrium w/ a solution to that of pure water The more ions (salts) or molecules (sugars) in solution the lower the water activity
Water Activity of Several Substances
Table 6.2
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
Effect of Sodium Chloride Concentrations on Growth
Figure 6.25

Halophiles: organisms that grow best at reduced water potential; have a specific requirement for NaCl (7-10%) 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 (1-3% NaCl)
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
Compatible Solutes of Microorganisms

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
Growth Versus Oxygen Concentration
Figure 6.27
Oxygen Relationships of Microorganisms

Special techniques are needed to grow aerobic and
anaerobic microorganisms
Reducing agents: chemicals that may be added to culture media to reduce oxygen (e.g., thioglycolate)
Incubation under Anoxic Conditions
Figure 6.28a
Incubation under Anoxic Conditions
Figure 6.28b

Several toxic forms of oxygen can be formed in the cell
Single oxygen
Superoxide anion Hydrogen peroxide Hydroxyl radical
Four-Electron Reduction of O2 to H2O
Figure 6.29

Enzymes are present to neutralize most of these toxic
oxygen species
Enzymes that Destroy Toxic Oxygen Species
Figure 6.30
Method for Testing a Microbial Culture for Catalase
Figure 6.31

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Brock Biology of Microorganisms

Madigan / Martinko Dunlap / Clark

I. Bacterial Cell Division

6.2 Fts Proteins and Cell Division

6.4 Peptidoglycan Synthesis and Cell Division

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.1 Cell Growth and Binary Fission

Animation: Binary Fission

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Binary Fission in a Rod-Shaped Prokaryote

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.3 Skip Section

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.4 Peptidoglycan Synthesis and Cell Division

Transpeptidation: final step in cell wall synthesis

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

II. Growth of Bacterial Populations

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.5 Growth Terminology and the Concept of Exponential Growth

Most bacteria have shorter generation times than

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.5 Growth Terminology and the Concept of Exponential Growth

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

The Rate of Growth of a Microbial Culture

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Calculating Microbial Growth Parameters

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.6 The Mathematics of Exponential Growth

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.6 The Mathematics of Exponential Growth

and n is the number of generations during the

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.7 The Microbial Growth Cycle

Animation: Bacterial Growth Curve

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Typical Growth Curve for a Bacterial Population

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.7 The Microbial Growth Cycle

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.7 The Microbial Growth Cycle

decrease in cell numbers

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

III. Measuring Microbial Growth

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.9 Measurements of Total Cell Numbers: Microscopic Counts

Small cells difficult to see and can be overlooked

6.9 Measurements of Total Cell Numbers: Microscopic Counts

Limitations of microscopic counts (contd)

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Direct Microscopic Counting Procedure

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.9 Measurements of Total Cell Numbers: Microscopic Counts

samples is with a flow cytometer

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

6.10 Viable Cell Counting

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Spread-Plate Method for the Viable Count

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Pour-Plate Method for the Viable Count

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Procedure for Viable Counting Using Serial Dilutions

Copyright 2009 Pearson Education Inc., publishing as Pearson Benjamin Cummings