Dynamics of Prokaryotic Growth

1
Dynamics of Prokaryotic Growth

• Chapter 4

2
Principles of Bacterial Growth

• Prokaryotic cells divide by binary fission
• One cell divides into two
• Two into four etc.
• Cell growth is exponential
• Doubling of population with each cell division
• Exponential growth has important health
  consequences
• Generation time
• Time it takes for population to double
• A.k.a doubling time
• Varies among species

3
Principles of Bacterial Growth

• Growth can be calculated
• Nt N0 x 2n
• (Nt ) number of cells in population
• (N0 ) original number of cells in the population
• (n) number of divisions
• Example
• N0 10 cells in original population
• n 12
• 4 hours assuming 20 minute generation time
• Nt 10 x 212
• Nt 10 x 4,096
• Nt 40,960

4
Bacterial Growth in Nature

• Conditions in nature have profound effect on
  microbial growth
• Cells sense changing environment
• Synthesize compounds useful for growth
• Cells produce multicellular associations to
  increase survivability
• Example
• Biofilms
• Slime layers

Biofilm layer
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Bacterial Growth in Nature

• Biofilm
• Formation begins with planktonic bacteria attach
  to surfaces
• Other bacteria attach and grow on initial layer
• Has characteristic architecture
• Contain open channels for movement of nutrient
  and waste
• Cells within biofilms can cause disease
• Treatment becomes difficult
• Architecture resist immune response and
  antimicrobials
• Bioremediation is beneficial use of biofilm

6
Bacterial Growth in Nature

• Interactions of mixed microbial communities
• Prokaryotes live in mixed communities
• Many interactions are cooperative
• Waste of one organism nutrient for another
• Some cells compete for nutrient
• Synthesize toxic substance to inhibit growth of
  competitors

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Obtaining Pure Culture

• Pure culture defined as population of cells
  derived from single cell
• All cells genetically identical
• Cells grown in pure culture to study functions of
  specific species
• Pure culture obtained using special techniques
• Aseptic technique
• Minimizes potential contamination
• Cells grown on culture media
• Can be broth (liquid) or solid form

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Obtaining Pure Culture

• Culture media can be liquid or solid
• Liquid is broth media
• Used for growing large numbers of bacteria
• Solid media is broth media with addition of agar
• Agar marine algae extract
• Liquefies at temperatures above 95C
• Solidifies at 45C
• Remains solid at room temperature and body
  temperature

• Bacteria grow in colonies on solid media surface
• All cells in colony descend from single cell
• Approximately 1 million cells produce 1 visible
  colony

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Obtaining Pure Culture

• Streak-plate method
• Simplest and most commonly used in bacterial
  isolation
• Object is to reduce number of cells being spread
• Solid surface dilution
• Each successive spread decreases number of cells
  per streak

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Bacterial Growth in Laboratory Conditions

• Cells in laboratory grown in closed or batch
  system
• No new input of nutrient and no release of waste
• Population of cells increase in predictable
  fashion
• Follows a pattern called growth curve

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Bacterial Growth in Laboratory Conditions

• The Growth Curve
• Characterized by five distinct stages
• Lag stage
• Exponential or log stage
• Stationary stage
• Death stage
• Phase of prolonged decline

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Bacterial Growth in Laboratory Conditions

• Lag phase
• Number of cells does not increase in number
• Cells prepare for growth
• tooling up
• Log phase
• Period of exponential growth
• Doubling of population with each generation
• Produce primary metabolites
• Compounds required for growth
• Cells enter late log phase
• Synthesize secondary metabolites
• Used to enhance survival
• Antibiotics

13
Bacterial Growth in Laboratory Conditions

• Stationary phase
• Overall population remains relatively stable
• Cells exhausted nutrients
• Cell growth cell death
• Dying cell supply metabolites for replicating
  cells
• Death phase
• Total number of viable cells decreases
• Decrease at constant rate
• Death is exponential
• Much slower rate than growth

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Bacterial Growth in Laboratory Conditions

• Phase of prolonged decline
• Once nearly 99 of all cells dead, remaining
  cells enter prolonged decline
• Marked by very gradual decrease in viable
  population
• Phase may last months or years
• Most fit cells survive
• Each new cell more fit that previous

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Bacterial Growth in Laboratory Conditions

• Colony growth on solid medium
• In colony, cells eventually compete for resources
• Cells grow exponentially and eventually compete
  for nutrients
• Position within colony determines resource
  availability
• Cells on edge of colony have little competition
  and significant oxygen stores
• Cells in the middle of colony have high cell
  density
• Leads to increased competition and decreased
  availability of oxygen

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Bacterial Growth in Laboratory Conditions

• Continuous culture
• Bacterial culture can be maintained
• Continuous exponential growth can be sustained by
  use of chemostat
• Continually drips fresh nutrients in
• Releases same amount of waste product

17
Environmental Factors on Growth

• As group, prokaryotes inhabit nearly all
  environments
• Some live in comfortable habitats
• Some live in harsh environments
• Most of these are termed extremophiles and belong
  to domain Archaea
• Major conditions that influence growth
• Temperature
• Oxygen
• pH
• Water availability

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Environmental Factors on Growth

• Temperature
• Each species has well defined temperature range
• Within range lies optimum growth temperature
• Prokaryotes divided into 5 categories

• Psychrophile
• Optimum temperature -5C to 15C
• Found in Arctic and Antarctic regions
• Psychrotroph
• 20C to 30C
• Important in food spoilage
• Mesophile
• 25C to 45C
• More common
• Disease causing
• Thermophiles
• 45C to 70C
• Common in hot springs
• Hyperthermophiles
• 70C to 110C
• Usually members of Archaea
• Found in hydrothermal vents

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Environmental Factors on Growth

• Oxygen
• Prokaryotes divided based on oxygen requirements
• Obligate aerobes
• Absolute requirement for oxygen
• Use for energy production
• Obligate anaerobes
• No multiplication in presence of oxygen
• May cause death
• Facultative anaerobes
• Grow better with oxygen
• Use fermentation in absence of oxygen
• Microaerophiles
• Require oxygen in lower concentrations
• Higher concentration inhibitory
• Aerotolerant anaerobes
• Indifferent to oxygen, grow with or without
• Does not use oxygen to produce energy

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Environmental Factors on Growth

• pH
• Bacteria survive within pH range
• Neutrophiles
• Multiply between pH of 5 to 8
• Maintain optimum near neutral
• Acidophiles
• Thrive at pH below 5.5
• Maintains neutral internal pH pumping out protons
  (H)
• Alkalophiles
• Grow at pH above 8.5
• Maintain neutral internal pH through sodium ion
  exchange
• Exchange sodium ion for external H

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Environmental Factors on Growth

• Water availability
• All microorganisms require water for growth
• Water not available in all environments
• In high salt environments
• Bacteria increase internal solute concentration
• Synthesize small organic molecules
• Osmotolerant bacteria tolerate high salt
  environments
• Bacteria that require high salt for cell growth
  termed halophiles

22
Nutritional Factors on Growth

• Growth of prokaryotes depends on nutritional
  factors as well as physical environment
• Main factors to be considered are
• Required elements
• Growth factors
• Energy sources
• Nutritional diversity

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Nutritional Factors on Growth

• Required elements
• Major elements
• Carbon, oxygen, hydrogen, nitrogen, sulfur,
  phosphorus, potassium, magnesium, calcium and
  iron
• Essential components for macromolecules
• Organisms classified based on carbon usage
• Heterotrophs
• Use organism carbon as nutrient source
• Autotrophs
• Use inorganic carbon (CO2) as carbon source
• Trace elements
• Cobalt, zinc, copper, molybdenum and manganese
• Required in minute amounts

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Nutritional Factors on Growth

• Growth factors
• Some bacteria cannot synthesize some cell
  constituents
• These must be added to growth environment
• Referred to as growth factors
• Organisms can display wide variety of factor
  requirements
• Some need very few while others require many
• These termed fastidious

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Nutritional Factors on Growth

• Energy Sources
• Organisms derive energy from sunlight or chemical
  compounds
• Phototrophs
• Derive energy from sunlight
• Chemotrophs
• Derive energy from chemical compounds
• Organisms often grouped according to energy source

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Nutritional Factors on Growth

• Nutritional Diversity
• Organisms thrive due to their ability to use
  diverse sources of carbon and energy
• Photoautotrouphs
• Use sunlight and atmospheric carbon (CO2) as
  carbon source
• Called primary producers (Plants)
• Chemolithoautotrophs
• A.k.a chemoautotrophs or chemolitotrophs
• Use inorganic carbon for energy and use CO2 as
  carbon source
• Photoheterotrophs
• Energy from sunlight, carbon from organic
  compounds
• Chemoorganoheterotrophs
• a.k.a chemoheterotrophs or chemoorganotrophs
• Use organic compounds for energy and carbon
  source
• Most common among humans and other animals

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Laboratory Cultivation

• Knowing environmental and nutritional factors
  makes it possible to cultivate organisms in the
  laboratory
• Organisms are grown on culture media
• Media is classified as complex media or
  chemically defined media

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Laboratory Cultivation

• Complex media
• Contains a variety of ingredients
• There is no exact chemical formula for
  ingredients
• Can be highly variable
• Examples include
• Nutrient broth
• Blood agar
• Chocolate agar

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Laboratory Cultivation

• Chemically defined media
• Composed of precise amounts of pure chemical
• Generally not practically for routine laboratory
  use
• Invaluable in research
• Each batch is chemically identical
• Does not introduce experimental variable

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Laboratory Cultivation

• Special types of culture media
• These are used to detect or isolate particular
  organisms
• Are divided into selective and differential media

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Laboratory Cultivation

• Selective media
• Inhibits the growth of unwanted organisms
• Allows only sought after organism to grow
• Example
• Thayer-Martin agar
• For isolation of Neisseria gonorrhoeae
• MacConkey agar
• For isolation of Gram-negative bacteria

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Laboratory Cultivation

• Differential media
• Contains substance that bacteria change in
  recognizable way
• Example
• Blood agar
• Certain bacteria produce hemolysin to break down
  RBC
• Hemolysis
• MacConkey agar
• Contains pH indicator to identify bacteria the
  produce acid

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Laboratory Cultivation

• Providing appropriate atmospheric conditions
• Bacteria can be grouped by oxygen requirement
• Capnophile
• Microaerophile
• Anaerobe

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Laboratory Cultivation

• Capnophile
• Require increased CO2
• Achieve higher CO2 concentrations via
• Candle jar
• CO2 incubator
• Microaerophile
• Require higher CO2 than capnophile
• Incubated in gastight jar
• Chemical packet generates hydrogen and CO2

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Laboratory Cultivation

• Anaerobe
• Die in the presence of oxygen
• Even if exposed for short periods of time
• Incubated in anaerobe jar
• Chemical reaction converts atmospheric oxygen to
  water
• Organisms must be able to tolerate oxygen for
  brief period
• Reducing agents in media achieve anaerobic
  environment
• Agents react with oxygen to eliminate it
• Sodium thioglycolate
• Anaerobic chamber also used for cultivation

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Detecting Bacterial Growth

• Variety of techniques to determine growth
• Number of cells
• Total mass
• Detection of cellular products

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Detecting Bacterial Growth

• Direct cell count
• Useful in determining total number of cells
• Does not distinguish between living and dead
  cells
• Methods include
• Direct microscopic count
• Use of cell counting instruments

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Detecting Bacterial Growth

• Direct microscopic count
• One of the most rapid methods
• Number is measured in a know volume
• Liquid dispensed in specialized slide
• Counting chamber
• Viewed under microscope
• Cells counted
• Limitation
• Must have at least 10 million cells per ml to
  gain accurate estimate

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Detecting Bacterial Growth

• Cell counting instruments
• Counts cells in suspension
• Cells pass counter in single file
• Instrument measure changes in environment
• Coulter counter
• Detects changes in electrical resistance
• Flow cytometer
• Measures laser light

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Detecting Bacterial Growth

• Viable cell count
• Used to quantify living cells
• Cells able to multiply
• Valuable in monitoring bacterial growth
• Often used when cell counts are too low for other
  methods
• Methods include
• Plate counts
• Membrane filtration
• Most probable numbers

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Detecting Bacterial Growth

• Plate counts
• Measures viable cells growing on solid culture
  media
• Count based on assumption the one cell gives rise
  to one colony
• Number of colonies number of cells in sample
• Ideal number to count
• Between 30 and 300 colonies
• Sample normally diluted in 10-fold increments
• Plate count methods
• pour-plates
• Spread-plates methods

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Detecting Bacterial Growth

• Membrane filtration
• Used with relatively low numbers
• Known volume of liquid passed through membrane
  filter
• Filter pore size retains organism
• Filter is placed on appropriate growth medium and
  incubated
• Cells are counted

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Detecting Bacterial Growth

• Most probable numbers (MPN)
• Statistical assay
• Series of dilution sets created
• Each set inoculated with 10-fold less sample than
  previous set
• Sets incubated and results noted
• Results compared to MPN table
• Table gives statistical estimation of cell
  concentration

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Detecting Bacterial Growth

• Biomass measurement
• Cell mass can be determined via
• Turbidity
• Total weight
• Amounts of cellular chemical constituents

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Detecting Bacterial Growth

• Turbidity
• Measures with spectrophotometer
• Measures light transmitted through sample
• Measurement is inversely proportional to cell
  concentration
• Must be used in conjunction with other test once
  to determine cell numbers
• Limitation
• Must have high number of cells

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Detecting Bacterial Growth

• Total Weight
• Tedious and time consuming
• Not routinely used
• Useful in measuring filamentous organisms
• Wet weight
• Cells centrifuged down and liquid growth medium
  removed
• Packed cells weighed
• Dry weight
• Packed cells allowed to dry at 100C 8 to 12
  hours
• Cells weighed

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Detecting Bacterial Growth

• Detecting cell products
• Acid production
• pH indicator detects acids that result from sugar
  breakdown
• Gas production
• Gas production monitored using Durham tube
• Tube traps gas produced by bacteria
• ATP
• Presence of ATP detected by use of luciferase
• Enzyme catalyzes ATP dependent reaction
• If reaction occurs ATP present ? bacteria present