Growth, Culturing and

Counting of Bacteria

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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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Indirect Measurements of Microbial Growth

 Multiple tube
MPN test.
 Count positive
tubes and
compare to
statistical
MPN table.

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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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Other Methods: Estimating Bacterial Numbers
by Indirect Methods

Turbidity

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Direct Measurements of Microbial Growth

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Direct Measurements of Microbial Growth

Direct microscopic count

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Measuring Microbial Growth
I. Direct methods
 Plate counts (Pour Plate and Spread Plate)
 Filtration
 Direct microscopic count
II. Indirect methods
 MPN (Turbidity, Gas)
 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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Psychrotrophs/Psychrophiles

 Grow between 0°C and 20-30°C

 Cause food spoilage

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Psychrotrophs/Psychrophiles

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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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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
 PO 3–
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4 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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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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Anaerobic Culture Methods
 Anaerobic
chamber

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Capnophiles Require High CO2

 Candle jar

 CO2-packet

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Selective Media
 Suppress unwanted
microbes and
encourage desired
microbes.

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Differential Media
 Make it easy to distinguish colonies of different
microbes.

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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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