Kul Pert Mikroba

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                Scope of lecture
   Cell Growth & Binary Fission
   Peptidoglycan Synthesis & Cell Division
   Population Growth
   The Growth Cycle
   Direct Measurements of Microbial Growth:
       Total and Viable Counts
   Indirect Measurements of Microbial Growth:
       Turbidity
   Growth kinetic
          Microbial Growth

 Increase in the number of cells
 Increase in microbial mass

 “Because individual cells grow larger only
 to divide into new individuals, microbial
 growth is defined not in terms of cell size
 but as the increase in the number of cells,
 which occurs by cell division."
Binary Fission

                 Note nascent septum forming
            Cell growth
 > 2000 chemical reactions
 Some involve energy transformation
 Other involve biosynthesis of
  Small molecules => polymers =>
  macromolecules => Cell structures
    The General process
 Duplication of DNA
 Elongation of cell
 Septum formation

    cell-partition, result of growth of
   plasma membrane & cell-wall in
   opposing direction
 Separation of two daughter cell
Cell growth
   In some sp, separation of two daughter cell is incomplete
       Linear chains: linked bacilli or cocci
       Tetrads: cuboidal groups of 4 cocci
       Sarcinae: groups of 8 cocci in a cubical packet
       Grapelike clusters: staphylococci


              Growth duration

    time require for a complete growth cycle is
     dependent on number of factors: nutritional &
     E. coli in the best nutritional conditions the time

     (generation time) is about 20 min
  Several proteins implicated in the cell-division process

Fts   proteins (filamentous temperature sensitive)
   Essential for normal cell-division & chromosome
  replication process in prokaryotes


  a     key protein in the group
  have     even been found in mitochondria & chloroplasts
cell division:

• division apparatus
  of Fts proteins
  including FtsZ
• FtsZ ring
  formation follows
  DNA replication
• FtsZ ring defines
  division plane
 Appearance & breakdown FtsZ ring
                                         FtsZ ring and cell division
 during E. coli cel-cycle
                                       Phase contrast
                                       Nucleoid stain
                                       cell stained w/ specific FtsZ reagent
                                       Combination nucleoid + FtsZ staining

   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 depolymerize => 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
    Peptidoglycan Synthesis and Cell
New  wall formed before cell-division
At Fts Z ring:

  Small openings in cell wall

     are created by autolysin
  (enzyme present in Divisome)
  New cell material is
  simultaneously added (by
  bactoprenol) across the opening
  Coordination is important so the
  cell does not leak (lysis)
         Peptidoglycan Synthesis

   Bactoprenol
      Lipid carrier molecule
      transports peptidoglycan building blocks across the membrane by rending
     precursor sufficiently hydrophobic
          bonds to N-acetyl (glucosamine / muramic acid) / pentapetide
         peptidoglycan precursor
          once in the periplasm:

         bactoprenol interacts w/ enzymes that insert cell-wall precursor into the
         growing point of the cell-wall & catalyze glycosidic bond formation
        Peptidoglycan Synthesis
Final   step in cell wall synthesis
Formation     of peptide cross-links
between muramic acid residues in
adjacent glycan chains
G-    diaminopimelic acid & D-Ala
G+ L-Lys     & D-Ala (interbridge)

   Penicillin-binding proteins: in periplasma of G-
When  penicillin binds to these proteins, no wall synthesis 
continuous action of autolysins weakens the cell wall  lysis
             Population Growth
• Increase in number of cells
• Increase in microbial mass

Growth rate (μ):
                                      Data for a population that doubles every 30 min.
Change in the number of cells/
unit of time

Generation (n):
Interval between two divisions

Generation time (g):
• Time for population to double   Data plotted on an arithmetic and a logarithmic scale
  during the exponential phase
• Time between two cell-           The rate of growth of a microbial culture
           Exponential Growth
   substrate and nutrients are abundant
   growth rate : proportional to the number of
          X           X = concentration of
     dt                      microorganisms at time t
    dX                   t = time
          dt           = proportionality constant or
                            specific growth rate, [time-1]
    ln(     )  t   dX/dt = microbial growth rate,
        X                    [mass/volume time]
    X  X e
                     Population Growth
   Exponential Growth:                       No = 5 x 107, Nt = 1 x 108, t = 2h
      Population doubles per unit of time    → n = 1 generation
                                              → g = 2/1 → g = 2 h
      # mo increases logarithmically:
      1 → 2 → 4 → 8 → 16 → 32…2n)                                         5 x 107
      mathematically exponential growth
    Nt = N0 2n => log Nt = log N0 + n log 2
    => n = 3.3 (log Nt - log N0 )                                                   g
      Nt = # cells at time t
      N0 = # initial cells
      n = # of generations during time (t)
                                              → g = 0.301/0.15 (slope) → g = 2 h
   Generation time (g):
      directly from graph                      Method of estimating the generation
      derived from n  g = t / n               times (g) of exponentially growing
      from the slope = 0.301/g                 populations with generation times of
                                                (a) 6 h and (b) 2 h from data plotted
   Growth rate constant (k):
                                                on semi logarithmic graphs.
      measure # generation /unit time
      k = ln 2 / g = 0.693 / g
 Substrate Limited Growth

     Ks  S

dX    m SX
dt   Ks  S

m = maximum specific growth rate [day-1]
 S = concentration of limiting substrate [mg/L]
Ks = Monod, or half-velocity constant [mg/L]
       Substrate Utilization
 Y 

Y = substrate yield,
   [mass of biomass/mass of substrate consumed]

 Y        dt
     dS       dS
 dS   1      m SX
         (        )
 dt   Y     Ks  S
              The Growth Cycle
Lag-phase: mo adjusts new environment, synthesizes enzymes &
essential constituents, repairs any lesions from earlier injury e.g.
freezing, drying, heating. No cell-division occur.

Exponential (Log) Growth Phase: Nt = N02n
generation time (or time to doubling cell-number) is constant
                The Growth Cycle
Stationary Phase:
Essential nutrient used up & waste & inhibitory products accumulate
Many cell-functions may continue
Reproduction (cell-division) & cell-death are balanced
(No net increase cell-number)

Death (decline) Phase:
When the death rate exceeds the rate of reproduction
Sometimes accompanied by cell-lysis
Exponential decline phase
        Direct Measurements of Microbial
              Growth: Total Counts
   Estimation of total cell mass/number is essential in most
    studies involving growth (measure growth rate,
    substrate utilization, effects of inhibitors as antibiotic)
   Methods to determine cell mass
     Direct (wet & dry weight)
     Indirect
            by chemical analysis of specific cellular component (nitrogen)
   Total number of mo determine
       Direct (direct counting or viability counting)
       Indirect (turbidity)
     Direct Measurements of Microbial
           Growth: Total Counts
        direct microscopic counting
              • using Petroff-Hausser counting chamber
              •quick way of estimating cell number
              •known volume of sample dried on slide & in counting chamber

              •Living & dead cells counted; Small cells difficult to see
              •Precision difficult to achieve
              •Phase contrast microscope required w/ no staining sample
              •Not suitable for cell-suspension at low cell density (sample
Direct Measurements of Microbial
     Growth: Viable Counts
 Surface drop (Miles-Misra): 20 μl

         ( 1 ml)

 Able to divide & form colony on suitable agar plate medium
 Each viable cell => one colony => CFU
Direct Measurements of Microbial
     Growth: Viable Counts
          Pour plate

                       Spread plates:
                       30-100 colonies

                       Pour plates:
                       30-300 colonies

                       Surface drop
                       10-30 colonies
   Direct Measurements of Microbial
        Growth: Viable Counts


In natural samples
Stimation of the total cells number

Direct microscopic counts >>> Viable counts (100-400 X)

In   microscopic counts dead & living cells are counted
In   viable counts only living cells
Every    viable counting method is selective
Direct Measurements of Microbial
     Growth: Viable Counts

                                       - High sensitivity
- The number of colonies depends on:
   Inoculum size                       - Could be made selective
   Inoculum conditions
   Selecting Culture medium
   Length of incubation
   Temperature of incubation

- Clumps of cell => 1 colony => CFU
       Indirect Measurements of
       Microbial Growth: Turbidity
 PHOTOMETER: Klett units
    - Simple filter generate light,
    relatively a narrow wavelength
Optical density (540, 600 or 660 nm)
    - Prisma of diffraction generate a
    very narrow band of wavelength
 Both measure unscattered light
 # Cells proportional Klett unit or
    OD except at high cell density
 Standard curve needed relating
    direct to indirect measurements
    - OD vs. # Cells (e.g. viable count)
    - OD vs. dry weight
 - Quick & easy
 - Do not disturb culture

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