Growth & Culturing

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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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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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 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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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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