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Chapter 6
Microbial Growth
© 2013 Pearson Education, Inc.
Copyright © 2013 Pearson Education, Inc.
Lectures prepared by Christine L. Case
Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc.
Microbial Growth
 Increase in number of cells, not cell size
 Populations
 Colonies
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The Requirements for Growth
 Physical requirements
 Temperature
 pH
 Osmotic pressure
 Chemical requirements





Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factor
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Physical Requirements
 Temperature
 Minimum growth temperature
 Optimum growth temperature
 Maximum growth temperature
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Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature.
Thermophiles
Hyperthermophiles
Mesophiles
Psychrotrophs
Psychrophiles
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Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent.
Water is being emitted through the ocean floor at temperatures above 100°C.
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Psychrotrophs
 Grow between 0°C and 20–30°C
 Cause food spoilage
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Figure 6.2 Food preservation temperatures.
Temperatures in this range destroy most microbes,
although lower temperatures take more time.
Very slow bacterial growth.
Danger zone
Rapid growth of bacteria; some may produce toxins.
Many bacteria survive; some may grow.
Refrigerator temperatures; may allow slow growth
of spoilage bacteria, very few pathogens.
No significant growth below freezing.
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Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.
15 cm (6′′) deep
5 cm (2′′) deep
Refrigerator air
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Approximate temperature
range at which Bacillus cereus
multiplies in rice
pH
 Most bacteria grow between pH 6.5 and 7.5
 Molds and yeasts grow between pH 5 and 6
 Acidophiles grow in acidic environments
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Osmotic Pressure
 Hypertonic environments, or an increase in salt or
sugar, cause plasmolysis
 Extreme or obligate halophiles require high
osmotic pressure
 Facultative halophiles tolerate high osmotic
pressure
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Figure 6.4 Plasmolysis.
Plasma
membrane
Cell wall
Plasma
membrane
H2O
Cytoplasm
NaCl 0.85%
Cell in isotonic solution.
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Cytoplasm
NaCl 10%
Plasmolyzed cell in hypertonic
solution.
Chemical Requirements
 Carbon
 Structural organic molecules, energy source
 Chemoheterotrophs use organic carbon sources
 Autotrophs use CO2
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Chemical Requirements
 Nitrogen




In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3–
A few bacteria use N2 in nitrogen fixation
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Chemical Requirements
 Sulfur
 In amino acids, thiamine, and biotin
 Most bacteria decompose proteins
 Some bacteria use SO42– or H2S
 Phosphorus
 In DNA, RNA, ATP, and membranes
 PO43– is a source of phosphorus
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Chemical Requirements
 Trace elements
 Inorganic elements required in small amounts
 Usually as enzyme cofactors
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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
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Toxic Oxygen
 Singlet oxygen: 1O2− boosted to a higher-energy
state
 Superoxide free radicals: O2
O2 + O2 + 2 H+
Superoxide dismutase
H 2 O2 + O 2
 Peroxide anion: O22–
2 H 2 O2
H 2 O2 + 2 H +
Catalase
Peroxidase
 Hydroxyl radical (OH•)
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2 H 2 O + O2
2 H2O
Organic Growth Factors
 Organic compounds obtained from the environment
 Vitamins, amino acids, purines, and pyrimidines
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Biofilms
 Microbial communities
 Form slime or hydrogels
 Bacteria attracted by chemicals via quorum sensing
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Figure 6.5 Biofilms.
Clumps of bacteria
adhering to surface
Surface
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Water currents
Migrating
clump of
bacteria
Biofilms
 Share nutrients
 Sheltered from harmful factors
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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.
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Biofilms
 Patients with indwelling catheters received
contaminated heparin
 Bacterial numbers in contaminated heparin were too
low to cause infection
 84–421 days after exposure, patients developed
infections
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Culture Media
 Culture medium: nutrients prepared for microbial
growth
 Sterile: no living microbes
 Inoculum: introduction of microbes into medium
 Culture: microbes growing in/on culture medium
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Agar
 Complex polysaccharide
 Used as solidifying agent for culture media in Petri
plates, slants, and deeps
 Generally not metabolized by microbes
 Liquefies at 100°C
 Solidifies at ~40°C
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Culture Media
 Chemically defined media: exact chemical
composition is known
 Complex media: extracts and digests of yeasts,
meat, or plants
 Nutrient broth
 Nutrient agar
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Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia
coli
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Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides
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Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria
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Anaerobic Culture Methods
 Reducing media
 Contain chemicals (thioglycolate or oxyrase) that
combine O2
 Heated to drive off O2
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Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Lid with
O-ring gasket
Clamp with
clamp screw
Envelope containing
sodium bicarbonate
and sodium
borohydride
Anaerobic indicator
(methylene blue)
Petri plates
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Palladium
catalyst pellets
Figure 6.7 An anaerobic chamber.
Air
lock
Arm
ports
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Capnophiles
 Microbes that require high CO2 conditions
 CO2 packet
 Candle jar
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Biosafety Levels
 BSL-1: no special precautions
 BSL-2: lab coat, gloves, eye protection
 BSL-3: biosafety cabinets to prevent airborne
transmission
 BSL-4: sealed, negative pressure
 Exhaust air is filtered twice
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Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.
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Differential Media
 Make it easy to distinguish colonies of different
microbes
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Figure 6.9 Blood agar, a differential medium containing red blood cells.
Bacterial
colonies
Hemolysis
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Selective Media
 Suppress unwanted microbes and encourage
desired microbes
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Figure 6.10 Differential medium.
Uninoculated
Staphylococcus
epidermis
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Staphylococcus
aureus
Enrichment Culture
 Encourages growth of desired microbe
 Assume a soil sample contains a few
phenol-degrading bacteria and thousands of
other bacteria
 Inoculate phenol-containing culture medium with the soil,
and incubate
 Transfer 1 ml to another flask of the phenol medium, and
incubate
 Transfer 1 ml to another flask of the phenol medium, and
incubate
 Only phenol-metabolizing bacteria will be growing
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Obtaining Pure Cultures
 A pure culture contains only one species or strain
 A colony is a population of cells arising from a
single cell or spore or from a group of attached cells
 A colony is often called a colony-forming unit
(CFU)
 The streak plate method is used to isolate pure
cultures
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Figure 6.11 The streak plate method for isolating pure bacterial cultures.
1
2
3
Colonies
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Preserving Bacterial Cultures
 Deep-freezing: –50° to –95°C
 Lyophilization (freeze-drying): frozen
(–54° to –72°C) and dehydrated in a vacuum
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Reproduction in Prokaryotes




Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
ANIMATION Bacterial Growth: Overview
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Figure 6.12a Binary fission in bacteria.
Cell elongates and
DNA is replicated.
Cell wall and plasma
membrane begin to constrict.
Cell wall
Plasma
membrane
DNA
(nucleoid)
Cross-wall forms,
completely separating
the two DNA copies.
Cells separate.
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(a) A diagram of the sequence of cell division
Figure 6.12b Binary fission in bacteria.
Partially formed cross-wall
DNA (nucleoid)
(b) A thin section of a cell of Bacillus licheniformis
starting to divide
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Cell wall
Figure 6.13a Cell division.
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Figure 6.13b Cell division.
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Generation Time
 If 100 cells growing for 5 hours produced
1,720,320 cells:
Number of generations =
Generation time =
Log number of cells (end) − Log number of cells (beginning)
0.301
60 min × hours
Number of generations
ANIMATION Binary Fission
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= 21 minutes/generation
Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed
line) and arithmetically (solid line).
Log10 = 4.52
Log10 = 3.01
Log10 = 1.51
(262,144)
(131,072)
(32)
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(524,288)
(65,536)
(32,768)
(1024)
Generations
Number of cells
Log10 of number of cells
(1,048,576)
Log10 = 6.02
Figure 6.15 Understanding the Bacterial Growth Curve.
Lag Phase
Log Phase
Stationary Phase
Death Phase
Intense activity
preparing for
population growth,
but no increase in
population.
Logarithmic, or
exponential,
increase in
population.
Period of equilibrium;
microbial deaths
balance production of
new cells.
Population Is
decreasing at a
logarithmic rate.
The logarithmic growth
in the log phase is due to
reproduction by binary
fission (bacteria) or
mitosis (yeast).
Staphylococcus spp.
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Figure 6.16 Serial dilutions and plate counts.
1 ml
1 ml
Original
inoculum
1 ml
1 ml
1 ml
9 m broth
in each tube
Dilutions
1:10
1 ml
1:100
1 ml
1:1000
1:10,000
1 ml
1 ml
1:100,000
1 ml
Plating
1:10
(10-1)
1:100
(10-2)
1:1000
(10-3)
1:10,000
(10-4)
1:100,000
(10-5)
Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml
(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)
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Plate Counts
 After incubation, count colonies on plates that have
25–250 colonies (CFUs)
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Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method
The spread plate method
Inoculate
1.0 or 0.1 ml
empty plate.
0.1 ml
Inoculate plate
containing
solid medium.
Bacterial
dilution
Add melted
nutrient
agar.
Spread inoculum
over surface
evenly.
Swirl to
mix.
Colonies
grow on
and in
solidified
medium.
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Colonies grow
only on surface
of medium.
Figure 6.18 Counting bacteria by filtration.
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Most Probable Number
 Multiple tube MPN test
 Count positive tubes
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Figure 6.19a The most probable number (MPN) method.
Volume of
Inoculum for
Each Set of
Five Tubes
(a) Most probable number (MPN) dilution series.
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Most Probable Number
 Compare with a statistical table
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Figure 6.19b The most probable number (MPN) method.
(b) MPN table.
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Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added here and fills the
shallow volume over the squares by capillary
action.
Bacterial
suspension
Microscopic count: All cells in
several large squares are
counted, and the numbers are
averaged. The large square shown
here has 14 bacterial cells.
Cover glass
Slide
Location of squares
Cross section of a cell counter.
The depth under the cover glass and the area
of the squares are known, so the volume of the
bacterial suspension over the squares can be
calculated (depth × area).
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The volume of fluid over the
large square is 1/1,250,000
of a milliliter. If it contains 14 cells,
as shown here, then
there are 14 × 1,250,000 =
17,500,000 cells in a milliliter.
Direct Microscopic Count
Number of cells counted
Number of bacteria/ml =
Volume of area counted
14
= 17,500,000
−7
8 × 10
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Figure 6.21 Turbidity estimation of bacterial numbers.
Light source
Spectrophotometer
Light
Scattered light
that does not
reach detector
Blank
Bacterial suspension
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Light-sensitive
detector
Measuring Microbial Growth
Direct Methods
 Plate counts
 Filtration
 MPN
 Direct microscopic count
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Indirect Methods
 Turbidity
 Metabolic activity
 Dry weight