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Microsoft PowerPoint - 08 Lecture 3 - microbial growth.ppt

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					                          Lecture 3

        Microbial growth – quantification &
                     kinetics
                     ki ti



                                                                            1




                               Aims
• Understand the concept of microbial growth
• Describe the steps involved in bacterial replication (binary fission)
  Differentiate b t
• Diff                   bacterial ll      th bacterial     li ti
          ti t between b t i l cell growth, b t i l replication and     d
  bacterial population growth
• Understand the calculation of population growth parameters
• Describe the bacterial population growth curve (batch culture)
• Describe what is happening to bacterial cells at each stage of the
  batch cycle growth cycle
• Describe commonly used methods for counting bacteria – direct
  microscopic count, viable count, turbidometric methods
                                              culture
• Differentiate batch culture from continuous culture.




                                                                            2




                                                                                1
                  Microbial growth
• Increase in number of bacterial cells rather
  than i        in i     f individual bacteria
  th increase i size of i di id l b t i
• Bacterial species only maintained if
  population continues to grow




                                             3




         Bacterial cell replication
• Bacterial cell is able to
  duplicate itself – asexual
  process – binary fission
• Involves many chemical
  reactions
    – Energy transformation
    – Biosynthesis of small
      molecules (enzymes, co-
      factors)
    – Polymerisation of
      macromolecules from
      monomers
• Assembly of macromolecules,
  formation of cellular structures
  (cell wall, cytoplasmic
  membrane ……….)


                                             4




                                                 2
                     Binary fission
• Prokaryotic cells divides into 2
  daughter cells – binary fission
  eg:
• Escherichia coli
   – Cells elongate to approx 2x
      length of smallest cell
   – Partition (septum) forms
      that eventually divides the
      cell into 2 daughter cells;
      partition is result of inward
      growth of cytoplasmic
      membrane and cell wall
      from opposite sides until
      they meet in the middle
   – Two daughter cells are           5
      pinched off




                     Binary fission
• During growth cycle all cellular
  components increase in
  number so each daughter cell
  receives a complete
  chromosome, macromolecules
  etc
• Note that DNA remains
  attached to cell membrane
  during division & replication of
  the chromosome so that as
  septum forms each daughter
  cell receives a complete
  chromosome copy
• Time required for a complete
  growth cycle is very variable –
  dependent on nutritional &
  genetic factors

                                      6




                                          3
                             Cell division
•   Specific proteins involved – form
    division apparatus (see Brock
    section 6.2)
               )
•   FtsZ proteins interact to form =>
    Divisome
     –    ring around middle cell (in yellow)
     –    DNA synthesis stop ⇒ Fts Z ring
         formation between 2 DNA
         molecules
•    FtsZ ring depolymerises =>
    inward growth of new membrane
    & wall material in both directions
    until a cell becomes twice its
    original length
•   Constriction: occurs to form 2-
    daughter cells




                                                                                      7




                     Population growth
• Growth rate = change in                                           Mean generation
  cell number or cell mass                                          time (hours)
         it ti
  per unit time
• Generation = formation of                     Bacillus megaterium 0.5
  2 daughter cells from one
                                                Escherichia coli    0.33
  cell
                                                Treponema pallidum 34
• Generation time = time
  for one generation to                         Saccharomyces       2
  occur; generation time =                      cerevisiae* (a
                                                fungus)
  doubling time
                                                Paramecium          10.5
• Note during one                               caudatum* (a
  generation both cell                          protozoa)

  number and cell mass                               * asexual reproduction           8
  double




                                                                                          4
Exponential (logarithmic) growth
• Pattern of population increase     time       Number of     Number of
                                                generations   organisms
  where the number of cells
  doubles in each generation         0          0             1
  time = exponential growth
• Note that in exponential growth
                                     3 hours    9             29 (512)
  the rate of increase in cell
  number is slow initially but
  increases at an ever faster rate   10 hours   30            230 (109)
  – in later stages there is an
  explosive increase in cell
  numbers                            48 hours   144           2144 (1043)




                                                                            9




              Growth parameters
• Increase in cell numbers in exponential growth –
  geometric progression of number 2
• 21→22 = increase from 2 to 4; 22→23 = increase from 4
  to 8 …..so a direct relationship between cells present at
  the start and cells present after a period of exponential
  growth:
    – N =N02n; N=final cell number, N0=initial cell number, n=number
      of generations
• If N and N0 are known, generation time can be
  calculated
• Growth rate constant (k) can also be calculated
• If n and t are known generation time and growth rate
  constant can be calculated for different organisms
  growing under different conditions
                                                                            10




                                                                                 5
 Exponential growth




                       11




Mean Generation Time
  and Growth Rate




                       12




                            6
             The Growth Cycle
• Exponential growth is only part of the story
  In       l    d     t  (batch lt )
• I an enclosed system (b t h culture) see
  several distinct phases:
  –   Lag phase
  –   Exponential growth
  –   Stationary phase
  –   Death phase
• Note – this describes phases of bacterial growth
  of a population of cells, not individual cells

                                                 13




Typical growth curve for a bacterial
            population




                                                 14




                                                      7
•   When microbes inoculated into
    fresh medium they do not start to
    grow immediately (lag phase)         Lag phase
•   Length of lag phase variable –
    depends on history of the culture
    and growth conditions
      – exponentially growing culture
        inoculated into same media,  ,
        same growth conditions – no
        lag phase
      – old culture, same media &
        conditions – lag phase
        because cells need to
        replenish essential
        constituents to start growth &
        cell division cycle
      – Cells damaged (heat,
                  ,               )
        radiation, toxic chemicals) -
        lag phase as cells repair
        damage
      – Cells transferred from rich
        medium to poor culture
        medium, lag phase as cells
        have to synthesise more
        enzymes etc to enable
        synthesis of macromolecules              15
        not present in poor culture
        medium.




                      Exponential phase
     • Each cell divides to form
       2 cells; 2 cells divide to
       form 4 cells ……
     • Rate of exponential
       growth influenced by
       environmental conditions
       (temperature,
       composition of culture
       medium) & genetic
       characteristics of
       organism

                                                 16




                                                      8
                   Stationary phase
•   In a batch culture exponential
    growth cannot occur indefinitely
      – Essential nutrients in medium
         used up and/or
      – Some waste product of the
         organism builds up to an
         inhibitory advice
      – Exponential growth ceases =
         stationary phase
•   In stationary phase – no net
    increase or decrease in cell
    number
•   Many cell f
    M                ti       ti
              ll functions continue –
    energy metabolism, biosynthesis
•   In some populations some slow
    growth may continue – some cells
    die and some grow – 2 processes
    balance out so no net change
    (cryptic growth)
                                        17




                        Death phase
• If incubation continues after
  stationary phase, cells may
  remain alive and continue to
  metabolise OR they may die =
  death phase
• In some cases cell death is
  accompanied by lysis
• Rate of cell death generally
  slower than that of exponential
  growth




                                        18




                                             9
   Direct Measurement of microbial
               growth
• Population growth is
  measured by:                         Direct microscopic count

    – Following changes in
      the number of cells
    – Weight of some
      component of cell
      mass eg protein
    – Total dry weight of
      cells



                                                                  19




                       Viable count
• Only counts living cells
• Viable cell = cell that is able to
  divide & form offspring
• Usual method – determine the
  number of cells in the sample
  able to form colonies on
  suitable agar medium = plate
  count or colony count
• Assumption:
   – Each viable cell can form
      one colony




                                                                  20




                                                                       10
        Turbidity measurements of
             microbial growth




                                    21




              Continuous culture
• Continuous cultures – flow
  system of constant volume
  where fresh medium is added
  continuously and spent culture
  medium removed continuously
  – at a constant rate.
• Once the system is in
  equilibrium, the cell number
  and nutrient status remain
  constant – steady state




                                    22




                                         11
Processes occurring in a chemostat


                       (X)




                μ
                                     23




                                          12

				
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