Microbial Growth

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• Microbial Growth 

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• I. The Growth Curve

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• Closed system Batch Culture
• Closed culture vessel
• One batch of culture medium
• Different from continuous culture (see below)
• Nutrients used up, culture eventually dies
• Four stages of bacteria growth in batch culture

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Lag Phase

• A period of apparent inactivity in which the
  cells are adapting to a new environment and
  preparing for reproductive growth, usually by
  synthesizing new cell components
• ATP
• Ribosomal proteins
• rRNA
• tRNA
• Co-factors
• Enzymes

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• Varies in length depending upon the condition of
  the microorganisms and the nature of the medium
• Assessment of medium Receptors
• DNA synthesized initiation of cell division

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Exponential phase (Log Phase)

• Optimal growth rate and cell division dependent
  on medium, O2, temperature, pH, genetic
  composition
• Regular, constant cell division (logarithmically)
• Smooth curve division not synchronous
• Most useful phase for biochemical, physiological
  and DNA replication studies
• Biotechnology applications competent cells
  uptake of plasmid DNA
• Late log optimal plasmid concentration
• The population is most uniform in terms of
  chemical and physical properties during this
  period

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Stationary Phase

• When the population reaches 109/ml (106 for
  protozoan and algal cultures), cell division
  cell death (stasis)
• Nutrients become scarce
• O2 is depleted
• Toxic waste accumulates
• The number of viable microorganisms remains
  constant either because metabolically active
  cells stop reproducing or because the
  reproductive rate is balanced by the rate of cell
  death

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Death Phase

• Viable cell mass decreases
• Often logarithmic
• Cells not viable when inoculated into fresh
  medium
• Cells have reached the carrying capacity of their
  environment

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• The mathematics of growth-microbial growth can be
  described by certain mathematical terms
• Mean generation (doubling) time (g) is the time
  required for the population to double
• Mean growth rate constant is the number of
  generations per unit time, often expressed as
  generations per hour

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• Generation times vary markedly with the species
  of microorganism and environmental conditions
• they can range from 10 minutes for a few bacteria
  to several days with some eukaryotic
  microorganisms
• Population size 2n where n the number of
  generations

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• II. Measurement of Microbial Growth

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Counting cells directly (live and dead)

• Petroff-Hausser Counting Chamber
• Slide with depressed etched grids (25 squares)
• Covered with a coverslip
• 25 squares (area) 1mm2
• Depth 0.02mm
• Volume 2 x 10-5 ml in 25 squares
• Determination of cell numbers
• 20 cells in one square x 25 squares/2 x 10-5 ml
  2.5 x 107 cells/ml

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• Electronic Counter
• Coulter counter
• Measures electrical resistance as cells pass
  single file through a thin stream
• RBC and WBC are counted
• Less accurate with small cells
• High interference
• Clumping

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Counting only live cells

• Plating techniques (spread plate, pour plate)
  using serial dilutions
• Colony forming units (CFU) usually arise from one
  organism (but may be several if clumpy)
• Membrane filtration assay
• Membrane traps bacterial on the surface
• Membrane transferred to an agar plate
• Colonies grow ? counted
• Can use selective media (e.g. Endo agar for
  coliform counts in contaminated water supplies)

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Measurement of cell mass

• Cell mass increases as cell number increases
• Dry weight measurements
• Growth, concentration, wash, dried, weighed
• Spectrophotometric determination
• Light is scattered and is proportional to cell
  number
• Linear relationship between absorbance and cell
  density
• Often written as transmittance (as absorbance
  increases, transmittance decreases)
• Requires cultures to be 107/ml and upwards
  (slight turbidity)

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• III. Continuous Culture Techniques

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• Used to maintain cells in the exponential growth
  phase at a constant biomass concentration for
  extended periods of time
• Conditions are met by continual provision of
  nutrients and removal of wastes OPEN SYSTEM
• Constant conditions are maintained

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• Chemostat
• A continuous culture device that maintains a
  constant growth rate by
• supplying a medium containing a limited amount of
  an essential nutrient at a fixed rate
• removing medium that contains microorganisms at
  the same rate
• As fresh media is added to the chamber, bacteria
  are removed
• Limiting nutrients control growth rates
• Cell density depends on nutrient concentration

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• Turbidostat
• A continuous culture device that regulates the
  flow rate of media through the vessel in order to
  maintain a predetermined turbidity or cell
  density
• There is no limiting nutrient
• Absorbance is measured by a photocell (optical
  sensing device)
• The number of cells in culture controls the flow
  rate and the rate of growth of culture adjusts to
  this flow rate

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• Balanced and Unbalanced Growth

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• Balanced (exponential) growth occurs when all
  cellular components are synthesized at constant
  rates relative to one another
• Unbalanced growth occurs when the rates of
  synthesis of some components change relative to
  the rates of synthesis of other components.
• This usually occurs when the environmental
  conditions change

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• IV. Environmental Factors Affecting Microbial
  Growth

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Solutes and Water Activity

• Osmotic concentrations affect microbes (e.g.
  plasmolysis in hypertonic solutions)
• Water activity (aw) measurement of availability
  of water in particular environments
• Aw Psolution/Pwater (P vapor pressure)
• inversely related to osmotic pressure
• If the solution has a high osmotic pressure (high
  extracellular solute concentration), then its Aw
  low

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• Energy is required by microbes to tolerate low aw
  because in order to keep water, solute
  concentration inside of cells must be kept high
• Osmotolerance
• S. aureus can tolerate up to 3M NaCl
• Archaebacteria halophiles tolerate 2.8-6.2M NaCl
  (Great Salt Lake, Dead Sea)
• Avoidance of plasmolysis

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pH (Log scale of 0 14 each pH unit 10x
change)

• pH is the negative logarithm of the hydrogen ion
  concentration

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• Acidophiles grow best between pH 0 and 5.5
• Neutrophiles grow best between pH 5.5 and 8.0
• Alkalophiles grow best between pH 8.5 and 11.5

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• Extreme alkalophiles grow best at pH 10.0 or
  higher
• Despite wide variations in habitat pH, the
  internal pH of most microorganisms is maintained
  near neutrality either by proton/ion exchange or
  by internal buffering
• Sudden pH changes can inactivate enzymes and
  damage PMs
• Reason for buffering culture medium, usually with
  a weak acid/conjugate base pair (e.g.
  KH2PO4/K2HPO4 monobasic potassium/dibasic
  potassium)

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Temperature

• Microorganisms are sensitive to temperature
  changes
• Usually unicellular and poikilothermic
• Enzymes have temperature optima
• If temperature is too high, proteins denature,
  including enzymes, carriers and structural
  components
• Temperature ranges are enormous (-20 to 100oC)

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• Organisms exhibit distinct cardinal temperatures
  (minimal, maximal, and optimal growth
  temperatures)
• If an organism has a limited growth temperature
  range stenothermal (e.g. N. gonorrhoeae)
• If an organism has a wide growth temperature
  range eurythermal (E. faecalis)

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• Psychrophiles can grow well at 0?C, have optimal
  growth at 15?C or lower, and usually will not
  grow above 20?C
• Arctic/Antarctic ocean
• Protein synthesis, enzymatic activity and
  transport systems have evolved to function at low
  temperatures
• Cell walls contain high levels of unsaturated
  fatty acids (semi-fluid when cold)

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• Psychrotrophs (facultative psychrophiles) can
  also grow at 0?C, but have growth optima between
  20?C and 30?C, and growth maxima at about 35?C
• Many are responsible for food spoilage in
  refrigerators
• Mesophiles have growth minima of 15 to 20?C,
  optima of 20 to 45?C, and maxima of about 45?C or
  lower
• Majority of human pathogens

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• Thermophiles have growth minima around 45?C, and
  optima of 55 to 65?C
• Hot springs, hot water pipes, compost heaps
• Lipids in PM more saturated than mesophiles
  (higher melting points)
• Hyperthermophiles have growth minima around 55?C
  and optima of 80 to 110?C
• Sea floor sulfur vents

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• Oxygen concentration
• Obligate aerobes are completely dependent on
  atmospheric O2 for growth
• Oxygen is used as the terminal electron acceptor
  for electron transport in aerobic respiration
• Facultative anaerobes do not require O2 for
  growth, but do grow better in its presence
• Aerotolerant anaerobes ignore O2 and grow equally
  well whether it is present or not

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• Obligate (strict) anaerobes do not tolerate O2
  and die in its presence
• Microaerophiles are damaged by the normal
  atmospheric level of O2 (20) but require lower
  levels (2 to 10) for growth

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• Oxygen tolerance is determined by an organisms
  ability to destroy toxic oxidizing products of
  oxygen reduction
• Remember, because oxygen has two unpaired outer
  orbital electrons, it accepts electrons readily

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• Toxic compounds
• Superoxide radical
• O2 e- ? O2-
• Hydrogen peroxide
• O2- e- 2H ? H2O2
• Hydroxyl radical
• H2O2 e- H ? H2O OH
• These compounds are used deliberately by
  phagocytic WBC to break down intracellular
  microbes (respiratory burst)

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• Solution used by obligate aerobes and facultative
  anaerobes
• Produce enzymes that convert these toxic
  oxidizing products to non-toxic compounds
• Superoxide dismutase
• 2O2- 2H ? O2 H2O2
• Catalase
• 2H2O2 ? 2H2O O2
• Aerotolerant microbes have SOD Obligate
  anaerobes lack SOD and catalase or have low
  concentrations

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• Laboratory considerations
• Aerobic cultures
• Shaken or sterile air introduced to medium

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• Anaerobic cultures
• Remove oxygen
• Include reducing agents in medium (e.g.
  thioglycollate or cysteine
• Dissolved oxygen is destroyed
• Growth beneath surface
• Replace oxygen with nitrogen gas and CO2 gas

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• Gas-Pak jar
• H2 palladium catalyst O2 ? H2O
• Bags or pouches ? CaCO3 ? CO2 rich atmosphere

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• Pressure
• Barotolerant organisms are adversely affected by
  increased pressure, but not as severely as are
  nontolerant organisms
• Barophilic organisms require, or grow more
  rapidly in the presence of, increased pressure

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• Radiation
• Ultraviolet radiation damages cells by causing
  the formation of thymine dimers in DNA
• Photoreactivation repairs thymine dimers by
  direct splitting when the cells are exposed to
  blue light
• Dark reactivation repairs thymine dimers by
  excision and replacement in the absence of light

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• Ionizing radiation such as X rays or gamma rays
  are even more harmful to microorganisms than
  ultraviolet radiation
• Low levels produce mutations and may indirectly
  result in death
• High levels are directly lethal by direct damage
  to cellular macromolecules or through the
  production of oxygen free radicals