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Microbial Growth

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Title: Microbial Growth


1
  • Microbial Growth 

2
  • I. The Growth Curve

3
  • 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

4
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

5
  • 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

6
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

7
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

8
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

9
  • 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

10
  • 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

11
  • II. Measurement of Microbial Growth

12
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

13
  • 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

14
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)

15
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)

16
  • III. Continuous Culture Techniques

17
  • 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

18
  • 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

19
  • 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

20
  • Balanced and Unbalanced Growth

21
  • 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

22
  • IV. Environmental Factors Affecting Microbial
    Growth

23
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

24
  • 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

25
pH (Log scale of 0 14 each pH unit 10x
change)
  • pH is the negative logarithm of the hydrogen ion
    concentration

26
  • 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

27
  • 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)

28
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)

29
  • 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)

30
  • 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)

31
  • 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

32
  • 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

33
  • 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

34
  • 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

35
  • 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

36
  • 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)

37
  • 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

38
  • Laboratory considerations
  • Aerobic cultures
  • Shaken or sterile air introduced to medium

39
  • 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

40
  • Gas-Pak jar
  • H2 palladium catalyst O2 ? H2O
  • Bags or pouches ? CaCO3 ? CO2 rich atmosphere

41
  • 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

42
  • 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

43
  • 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
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