Dynamics of Microbial Growth
Chapter 4
Obtaining a Pure Culture
• Streak-plate method
• Simplest, most commonly used method for isolating
• Reduces number of cells with each series of streaks
Obtaining a Pure Culture
• Streak-plate method
• Simplest, most commonly used method for isolating
• Reduces number of cells with each series of streaks
• Separated cells form distinct, isolated colonies
Maintaining Stock Cultures
• A pure culture can be maintained as
stock culture
• Often stored in refrigerator as agar
slant
• Cells can be frozen at -70 degrees
Celsius for long-term storage
• Mixed with glycerol to prevent ice crystal
formation
• Can be freeze-dried
The Growth Curve
• Closed system: culture in which nutrients are not added, nor are wastes
removed
• Includes microorganisms grown on agar plates or in tubes or flasks of broth
• Also called batch cultures
Characteristic growth curve seen in closed broth culture
The Growth Curve
• Lag phase
• Number of cells does not increase
• Cells begin synthesizing enzymes required for growth
• Delay depends on conditions
• Exponential (log) phase
• Cells divide at constant rate
• Generation time measured
• Most sensitive to antibiotics
• Produce primary metabolites such as amino acids
• Secondary metabolites such as antibiotics are
produced as nutrients are depleted and wastes
accumulate
The Growth Curve
• Stationary phase
• Nutrient levels too low to sustain growth
• Total numbers remain constant
• Some die, releasing nutrients; others grow
• Continue to produce secondary metabolites
• Death phase
• Total number of viable cells decreases
• Cells die at constant rate
• Exponential, but usually much slower than
cell growth
• Phase of prolonged decline
• Some fraction may survive
• Adapted to tolerate worsened conditions
Colony Growth
• Growth of bacteria on solid medium and liquid
broth is similar, but there are important
differences.
• Position of single cell in a colony determines its
environment.
• Edge of colony
• Little competition for O2, nutrients
• Center of colony
• Depleted O2, nutrients
• Accumulation of potentially toxic wastes
• Cells at edges of colony may show exponential growth
while those in the center are in the death phase
Continuous Culture
Open system: culture to which nutrients are
continually added and waste products removed
• A chemostat provides an open system
that can maintain continuous growth
• Nutrient content and speed of addition can
be controlled to achieve constant growth
rate and cell density
• Produces relatively uniform population to
study response to different conditions
• Can maintain cells in log phase of growth to
harvest commercially valuable products
Environmental Factors That
Influence Microbial Growth
• As a group, microorganisms inhabit nearly all
environments
• Some live in comfortable habitats favored by humans
• Extremophiles live in harsh environments; most are archaea
• Major factors that affect microbial growth
• Temperature
• Atmosphere
• pH
• Water availability
Temperature Requirements
• Each species has well-defined temperature range
• Optimum growth usually close to upper end of range
• Psychrophiles: -5 degrees to 15 degrees Celsius
• Found in Arctic and Antarctic regions
• Psychrotrophs: 15 degrees to 30 degrees Celsius
• Important in spoilage of refrigerated foods
• Mesophiles: 25 degrees to 45 degrees Celsius
• Pathogens 35 degrees to 40 degrees Celsius
• Thermophiles: 45 degrees to 70 degrees Celsius
• Common in hot springs and compost heaps
• Hyperthermophiles: 70 degrees or greater
• Usually archaea
• Found in hydrothermal vents.
Oxygen (𝐎𝟐) Requirements
• Measure in shake tube:
• Boil nutrient agar to drive off O2; cool to just above solidifying
temperature; add microorganisms; gently swirl
• Solidified agar slows gas diffusion
• Top of tube is aerobic - O2 is present
• Bottom of tube is anaerobic - O2 is absent
• Position of growth indicates organism’s O 2 requirements
• Obligate aerobes: require O2
• Facultative aerobes: use O2, but don’t require it
• Obligate anaerobes: cannot use O2
• Microaerophiles: require small amounts of O2 only
• Aerotolerant anaerobes: obligate fermenters (can grow in O2, but they
don’t use it)
Oxygen (𝐎𝟐) Requirements
Obligate Facultative Obligate Microaerophil Aerotolerant
aerobe anaerobe anaerobe e anaerobe
Pattern of Growth
in a Shake Tube
Growth characteristics Grows only Grows best when Cannot grow Grows only if Grows equally
when O2 is O2 is available, but when O2 is small amounts well with or
available. also grows without present. of O2 are without O2.
it. available.
Use of O2 in energy- Requires O2 Uses O2 for Does not use Requires O2 Does not use
harvesting processes for respiration. respiration, if O2 . for respiration. O2 .
available.
Typical mechanisms Produces Produces Does not Produces Produces
to protect against superoxide superoxide produce some superoxide
reactive oxygen dismutase dismutase and superoxide superoxide dismutase but
species and catalase. catalase. dismutase or dismutase and not catalase.
catalase. catalase.
pH
• Bacteria survive a range of pH; have optimum
• Most maintain constant internal pH, typically near neutral
• Pump out protons H+ if in acidic environment
• Bring in protons H+ if in alkaline environment
• Most microbes are neutrophiles
• Range of pH 5 to 8; optimum near pH 7
• Food can be preserved by increasing acidity
• H. pylori grows in stomach; produces urease to split urea into CO 2 and ammonia
to decrease acidity of surroundings
• Acidophiles grow optimally at pH below 5.5
• Picrophilus oshimae has optimum pH of less than 1!
• Alkaliphiles grow optimally at pH above 8.5
Water Availability
• All microorganisms require water for growth
• Dissolved salts, sugars make water unavailable to cell
• If solute concentration is higher outside of cell, water diffuses out (osmosis)
• Salt, sugar used to preserve food
• Some microbes withstand or even require high salt
• Halotolerant: withstand up to 10% NaCl
• Staphylococcus on dry salty skin
• Halophiles: require high salt levels
• Marine bacteria approximately 3% NaCl
• Extreme halophiles
(Dead Sea, Utah’s salt flats)
Practice Time!
Categorize the following microbe and make sure to use the
correct terminology:
• Clostridium paradoxum grows optimally at 55℃, pH 9.3; it will not grow
in the presence of O2
Nutritional Factors That
Influence Microbial Growth
Chemical Function
Carbon, oxygen, and Component of amino acids, lipids, nucleic acids,
hydrogen and sugars
Nitrogen Component of amino acids and nucleic acids
Sulfur Component of some amino acids
Phosphorus Component of nucleic acids, membrane lipids, and ATP
Potassium, Required for the functioning of certain enzymes;
magnesium, additional functions as well
and calcium
Iron Part of certain enzymes
Nutritional Factors That
Influence Microbial Growth
• Growth factors: organic molecules that an organism cannot synthesize;
must be present in the environment
• Energy sources
• Sunlight, chemical compounds
• Phototrophs obtain energy from sunlight
• Plants, algae, photosynthetic bacteria
• Chemotrophs extract energy from chemicals
• Mammalian cells, fungi, many types of prokaryotes extract energy from organic
molecules
• Sugars, amino acids, fatty acids are common sources
• Some prokaryotes extract energy from inorganic chemicals
• Hydrogen sulfide, hydrogen gas
Energy and Carbon Sources
Used by Different Groups of
Microorganisms
Cultivating Microorganisms in
The Laboratory
• Requires a suitable growth medium and
an appropriate atmosphere.
• General Categories of Culture Media
• Complex media contain a variety of
ingredients
• Exact composition is highly variable
• Chemically defined media composed of
exact amounts of pure chemicals
• Typically slower growth as cells must synthesize
components
Special Types of Culture Media
• Selective media inhibit growth of certain species in a mixed
sample, while allowing growth of species of interest
• MacConkey agar is selective for Gram-negative rods
• Contains crystal violet that inhibits Gram-positive bacteria and bile salts that
inhibit most non-intestinal bacteria
Special Types of Culture Media
• Differential media contain substance that microbes change in
identifiable way
• Blood agar is differential
• Beta hemolysis produces clear zone
• Alpha hemolysis produces zone of greenish partial clearing
Special Types of Culture Media
• Selective or differential media can be used to detect or isolate a
species from a mixed population.
Selective and Differential Media
Methods to Detect & Measure
Microbial Growth: Direct Cell
Counts
• Direct cell counts: total numbers (living plus dead)
• Direct microscopic count
• Cell-counting instruments (Coulter counter, flow cytometer)
Flow Cytometry Animation
Methods to Detect & Measure
Microbial Growth: Viable Cell
Counts
• Viable cell counts: cells capable of
multiplying
• Can use selective, differential media
for particular species
• Plate counts: single cell gives rise to
colony
• Number of colonies reflects how many
cells were in sample
• Plate dilution series to obtain 30 to
300 colonies
Viable Cell Count: Serial Dilution
Animation
Methods to Detect & Measure
Microbial Growth: Spread/Pour
plate method
• Samples added to agar plate
• Spread plate method
• Pour plate methods
• Plate counts determine colony-
forming units (CFUs)
• Cells often attach to one another
and form a single colony
Methods to Detect & Measure
Microbial Growth: Membrane
Filteration
• Membrane filtration
• Known volume passed through sterile filter
• Captures and concentrates microbes by filtration
• Filter is incubated on appropriate agar medium
Methods to Detect & Measure
Microbial Growth: Biomass
• Measuring biomass:
measure cell mass
instead of number of
cells
• Turbidity of microbial
suspension is proportional
to concentration of cells
• Measured with
spectrophotometer
Using Spectrophotometer in
Microbiology Lab
