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Growth & Culturing
of Bacteria
Microbiology 130
Chapter 6
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Microbial Growth and Cell Division
 Microbial Growth Defined:
 Microbial growth is the increase in number of cells,
not cell size
 Mother cells
 Daughter cells
 Cell Division:
 Binary fission
 Tetrads
 Sarcinae
 Budding
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Reproduction in Prokaryotes
 Binary fission
 Budding
 Conidiospores (actinomycetes)
 Fragmentation of filaments
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Binary Fission
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Phases of Growth
 4 Phases:
 1) Lag phase 2) Log (logarithmic) phase
 3) Stationary phase
 4) Decline phase or death phase
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Log Phase
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Log Phase with Calculations
 If 100 cells growing for 5 hours produced 1,720,320
cells:
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Log phase
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4 Phases of Microbial Growth
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Growth in Colonies
 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).
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Measuring Bacterial Growth
Serial Dilutions
 Direct Measurements of Microbial Growth
 Plate counts: Perform serial dilutions of a sample
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Standard Plate Count
Inoculate
Petri plates
from serial
dilutions
2 methods:
Pour Plate
Spread Plate
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Plate Count
After incubation, count
colonies on plates that have
25-250 colonies (CFUs)
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Direct Measurements of Microbial Growth
Filtration
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Direct Measurements of Microbial Growth
 Multiple tube
MPN test.
 Count positive
tubes and
compare to
statistical
MPN table.
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Direct Measurements of Microbial Growth
Direct microscopic count
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Most Probable Number
 Estimate of number
 MPN 5 test tubes 10, 1, and 0.1 ml
 Growth is displayed by production of gas bubbles or by
becoming cloudy
 Estimates are from a known chart ( table 6.1 pg 149)
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Direct Measurements of Microbial Growth
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Other Methods: Estimating Bacterial Numbers
by Indirect Methods
Turbidity
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Measuring Microbial Growth
Direct methods
 Plate counts
 Filtration
 MPN
 Direct microscopic count
 Dry weight
Indirect methods
 Turbidity
 Metabolic activity
 Dry weight
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Factors Affecting Bacterial Growth
The Requirements for Growth:
Physical Requirements
 Temperature
 Minimum growth temperature
 Optimum growth temperature
 Maximum growth temperature
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Temperature
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Figure 6.1
Psychrotrophs/Psychrophiles
 Grow between 0°C and 20-30°C
 Cause food spoilage
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Psychrotrophs/Psychrophiles
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Figure 6.2
The Requirements for Growth:
Physical Requirements
 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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Mesophiles & Thermophiles
 Mesophiles grow best between 25 degrees C and 40 degrees C
 Thermophiles Heat loving- grow best at 50 - 60 degrees C
 Obligate thermophiles
 Facultative thermophiles-
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The Requirements for Growth:
Physical Factors - Chemical Requirements
Oxygen (O2)
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Toxic Forms of Oxygen
 Singlet oxygen: O2 boosted to a higher-energy state
 Superoxide free radicals: O2–
 Peroxide anion: O22–
 Hydroxyl radical (OH)
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The Requirements for Growth:
Physical Factors / Requirements
 Osmotic pressure
 Hypertonic environments, increase salt or sugar,
cause plasmolysis
 Extreme or obligate halophiles require high osmotic
pressure
 Facultative halophiles tolerate high osmotic pressure
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The Requirements for Growth:
Physical Requirements
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Figure 6.4
The Physical Requirements for Growth
 Moisture Hydrostatic Pressure Radiation-
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The Requirements for Growth:
Nutritional Factors - Chemical Requirements
 Carbon
 Structural organic molecules, energy source
 Chemoheterotrophs use organic carbon sources
 Autotrophs use CO2
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The Requirements for Growth:
Nutritional Factors - 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
 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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The Requirements for Growth:
Nutritional Factors - Chemical Requirements
 Trace elements
 Inorganic elements required in small amounts
 Usually as enzyme cofactors
 Vitamins- organic substances and growth factors
 Organic compounds obtained from the environment
 Vitamins, amino acids, purines, and pyrimidines
 Nutritional Complexity
 Locations of Enzymes
 Adaptations to Limited Nutrients
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Toxic Forms of Oxygen
 Singlet oxygen: O2 boosted to a higher-energy state
 Superoxide free radicals: O2–
 Peroxide anion: O22–
 Hydroxyl radical (OH)
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Sporulation / Endospores
 Formation of endospores in Bacillus, Clostridium
 and G+ genera
 Can Survive long periods of drought
 Axial nucleoid
 Endospore septum grows
 Spore coat and Exosporium
 Germination- 3 stages:
 1) Activation, 2) germination, 3) outgrowth
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Culturing Bacteria
 Methods of Obtaining Pure Culture
 1) The Streak Plate Method – sterile wire loop and
streaked in different patterns on agar
 2) Pour Plate Method- serial dilutions using melted
agar
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Streak Plate
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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
 Synthetic media
 Defined synthetic media
 Complex media
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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 ~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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Culture Media
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Tables 6.2, 6.4
Anaerobic Culture Methods
 Reducing media
 Contain chemicals (thioglycollate or oxyrase) that
combine O2
 Heated to drive off O2
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Anaerobic Culture Methods
 Anaerobic
jar
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Figure 6.5
Anaerobic Culture Methods
 Anaerobic
chamber
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Figure 6.6
Capnophiles Require High CO2
 Candle jar
 CO2-packet
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Figure 6.7
Selective Media
 Suppress unwanted
microbes and
encourage desired
microbes.
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Figure 6.9b–c
Differential Media
 Make it easy to distinguish colonies of different
microbes.
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Figure 6.9a
Enrichment Media
 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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Preserving Bacteria Cultures
 Deep-freezing: –50°to –95°C
 Lyophilization (freeze-drying): Frozen (–54° to –72°C)
and dehydrated in a vacuum
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Methods of Performing Multiple
Diagnostic Tests
 Enterotube Multitest API- simultaneous testing
 To identify enteric bacteria- cause food poisoning,
typhoid, shigellosis, gastroenteritis etc
 Living, But Non-culturable organisms:
 Some microbs can be observed with microscope,
identify their DNA but they can not be cultured
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