Download Dynamics of Prokaryotic Growth

Dynamics of Prokaryotic
Growth
Chapter 4
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Principles of bacteria growth.
Bacteria growth in nature.
Bacteria growth in laboratory.
Factors affect bacteria growth.
Detecting bacteria growth
Principles of Bacterial Growth
• Prokaryotic cells divide by binary
fission
– One cell divides into two
– Cell growth is exponential
• population double with each cell
division.
• Cell divide at constant pace
– Generation time
• Time it takes for population to double
• A.k.a doubling time
• Varies among species
Practical Problem
• 100 E. coli in potato salad.
• How many bacteria are there in salad 2
hrs later (if the double time is 20 min)?
• Why do we need to store the salad in
cooler?
Principles of Bacterial Growth
• Growth can be calculated
– Nt = N0 x 2n
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(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
Bacterial Growth in Nature
• Conditions in nature
have profound effect on
microbial growth
– Synthesize
compounds useful for
growth
– produce multicellular
associations to
increase survivability
Biofilm layer
• Biofilms: a community
formed by a group of
bacteria and their secreted
slimes.
Biofilm
– Can cause disease
• Difficult to kill
– Architecture resist
immune response
and antimicrobial
drugs
– Can be beneficial
• biofilm
Bacterial Growth in Nature
– Prokaryotes live in mixed communities
• Many interactions are cooperative
• Some cells compete for nutrient
Bacteria growth in Laboratory
• Culture media
– broth media
– Solid media is broth media
with addition of agar
• Agar marine algae extract
• Liquefies above 95°C
• Solidifies at 45°C
– Remains solid at room
temperature and body
temperature
– Bacteria grow in colonies
on solid media surface
• Single colony
Laboratory Cultivation
• Special types of culture media
– These are used to detect or isolate particular
organisms
– Are divided into selective and differential
media
Laboratory Cultivation
• Selective media
– Inhibits the growth of unwanted organisms
• Allows only sought after organism to grow
– Example
• Thayer-Martin agar (multiple antimicrobial)
– For isolation of Neisseria gonorrhoeae
• MacConkey agar (antimicrobial+bile salt)
– For isolation of Gram-negative intestinal bacteria
Laboratory Cultivation
• Differential media
– Contains substance
that bacteria change in
recognizable way
– Example
• Blood agar
– Test for hemolysis
• MacConkey agar
– pH indicator
Obtaining Pure Culture
• Pure culture is defined as population of
cells derived from single cell
– All cells are genetically identical
• to study functions of specific species
• Obtain pure culture
– Aseptic technique
Obtaining Pure Culture
• Streak-plate method
– Simplest and most
commonly used in
bacterial isolation
– Object is to reduce
number of cells being
spread
Bacterial Growth in
Laboratory Conditions
• Cells in laboratory grown in closed or
batch system
• Population of cells increase in predictable
fashion
– Follows a pattern called growth curve
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
Bacterial Growth in
Laboratory Conditions
Lag phase
– synthesis of cell components and
prepare for division
• Log phase
– exponential growth
• Cell divide at constant rate
– Produce primary metabolites
• Compounds required for growth
– Cells enter late log phase
• Cell wall and cell membrane
component changes
• Synthesize secondary metabolites
– Used to enhance survival
– Antibiotics
Bacterial Growth in Labortory Conditions
• Stationary phase
– Overall population remains
relatively stable
• Cells exhausted nutrients and
build up toxic waste
• Cell growth = cell death
• Death phase
– Total number of viable cells
decreases
• 99% of cells die at constant rate
– Death is exponential
• Much slower rate than growth
Bacterial Growth in
Laboratory Conditions
• Phase of 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
Bacterial Growth in
Laboratory Conditions
• Continuous culture
– Bacterial culture can be maintained
• Continuous exponential growth can be sustained
by use of chemostat
Bacterial Growth in
Laboratory Conditions
• Colony growth on solid medium
– 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
Questions
• What is biofilm?
• What is pure culture?
• What are the different stages of bacterial
growth?
• Selective and differential medium.
Environmental Factors on Growth
• Major conditions that influence growth
Temperature
Oxygen
pH
Water availability
Environmental Factors on Growth
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Psychrophile
– Optimum temperature -5°C to
15°C
• Found in Arctic and Antarctic
regions
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Psychrotroph
– 20°C to 30°C
• Important in food spoilage
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Mesophile
– 25°C to 45°C
• More common
• Disease causing
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Thermophiles
– 45°C to 70°C
• Common in hot springs
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Hyperthermophiles
– 70°C to 110°C
• Usually members of Archaea
• Found in hydrothermal vents
Environmental Factors on Growth
• Temperature and food storage
– 4C can slow down bacteria growth
– Freezing can stop bacteria growth
• Temperature and disease
– Different pathogen can only grow in different
part of body.
• Hansen’s disease
• Syphilis disease
Environmental Factors on Growth
• Oxygen
– Prokaryotes divided based on oxygen
requirements
• Obligate aerobes
– Absolute requirement for oxygen
» Use for energy production
» Micrococcus
• Obligate anaerobes
– No multiplication in presence of oxygen
» May cause death
» Clostridium
Environmental Factors on Growth
• Facultative anaerobes
– Grow better with oxygen
» Use fermentation in absence of oxygen
» E coil
• Microaerophiles
– Require oxygen in lower concentrations
» Higher concentration inhibitory
» Helicobacter pylori
• Aerotolerant anaerobes
– Indifferent to oxygen, grow with or without
» Does not use oxygen to produce energy
» Streptococcus
Environmental Factors - O2 availability
Decreasing
O2
Environmental Factors - O2 availability
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+
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
Nutritional Factors on Growth
• Growth of prokaryotes depends on
nutritional factors as well as physical
environment
• Main factors to be considered are:
– Required elements
– Energy sources
– Growth factors
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 organic carbon as carbon source
– Autotrophs
» Use inorganic carbon (CO2) as carbon source
– Trace elements
• Cobalt, zinc, copper, molybdenum and manganese
– Required in minute amounts
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
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
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
Questions
• Major factors that affect bacteria growth
• Growth factor
• Carbon source and energy source of
chemoheterotroph
Detecting Bacterial Growth
• Variety of techniques to determine growth
– Number of cells
– Total mass
– Detection of cellular products
Detecting Bacterial Growth
• Direct cell count
• Plate count
Detecting Bacterial Growth
• Direct microscopic count
– 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
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