MICROBIAL GROWTH

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• MICROBIAL GROWTH

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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

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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."
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Binary Fission
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• Cell growth
• gt 2000 chemical reactions
• Some involve energy transformation
• Other involve biosynthesis of
• Small molecules gt polymers gt
  macromolecules gt 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

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• 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

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Diplococcus
Streptococcus
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Tetrad
Sarcinae
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Staphylococcus
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Growth duration

• time require for a complete growth cycle is
  variable,
• dependent on number of factors nutritional
  genetic
• E. coli in the best nutritional conditions the
  time
• (generation time) is about 20 min

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• Several proteins implicated in the cell-division
  process
• Fts proteins (filamentous temperature sensitive)
• Essential for normal cell-division chromosome
  replication process in prokaryotes
• FtsZ
• a key protein in the group
• have even been found in mitochondria
  chloroplasts

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cell division

• divisome
• division apparatus of Fts proteins including FtsZ
• FtsZ ring formation follows DNA replication
• FtsZ ring defines division plane

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FtsZ ring and cell division
Appearance breakdown FtsZ ring during E. coli
cel-cycle
Phase contrast Nucleoid stain cell stained w/
specific FtsZ reagent Combination nucleoid FtsZ
staining

• FtsZ proteins interact to form gt Divisome
• ring around middle cell (in yellow)
• DNA synthesis stop ? Fts Z ring formation
  between 2 DNA molecules
• FtsZ ring depolymerize gt 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

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Peptidoglycan Synthesis and Cell Division

• 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)

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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

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Peptidoglycan Synthesis

• Transpeptidation
• 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

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Population Growth

• Growth
• Increase in number of cells
• Increase in microbial mass
• Growth rate (µ)
• Change in the number of cells/ unit of time
• Generation (n)
• Interval between two divisions
• Generation time (g)
• Time for population to double
• during the exponential phase
• Time between two cell- divisions

Data for a population that doubles every 30 min.
Data plotted on an arithmetic and a logarithmic
scale
The rate of growth of a microbial culture
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Exponential Growth

• substrate and nutrients are abundant
• growth rate proportional to the number of
  microorganisms

• X concentration of
• microorganisms at time t
• t time
• ? proportionality constant or
• specific growth rate, time-1
• dX/dt microbial growth rate,
• mass/volume time

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Population Growth

• Exponential Growth
• Population doubles per unit of time
• mo increases logarithmically
• 1 ? 2 ? 4 ? 8 ? 16 ? 322n) mathematically
  exponential growth
• Nt N0 2n gt log Nt log N0 n log 2
• gt n 3.3 (log Nt - log N0 )
• Nt cells at time t
• N0 initial cells
• n of generations during time (t)
• Generation time (g)
• directly from graph
• derived from n ? g t / n
• from the slope 0.301/g
• Growth rate constant (k)
• measure generation /unit time
• k ln 2 / g 0.693 / g

No 5 x 107, Nt 1 x 108, t 2h ? n 1
generation ? g 2/1 ? g 2 h
5 x 107
g
? g 0.301/0.15 (slope) ? g 2 h
Method of estimating the generation times (g) of
exponentially growing populations with generation
times of (a) 6 h and (b) 2 h from data plotted on
semi logarithmic graphs.
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Substrate Limited Growth
?m maximum specific growth rate day-1 S
concentration of limiting substrate mg/L Ks
Monod, or half-velocity constant mg/L
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Substrate Utilization
Y substrate yield, mass of biomass/mass
of substrate consumed
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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
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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
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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)

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Direct Measurements of Microbial Growth Total
Countsdirect 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 concentration)
• Motile cells has to be immobilized

Limitation
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Direct Measurements of Microbial Growth Viable
Counts
Surface drop (Miles-Misra) 20 µl
(? 1 ml)
VIABLE CELL Able to divide form colony on
suitable agar plate medium Each viable cell gt
one colony gt CFU
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Direct Measurements of Microbial Growth Viable
Counts
Pour plate
Spread plates 30-100 colonies Pour plates
30-300 colonies Surface drop (Miles-Misra)
10-30 colonies
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Direct Measurements of Microbial Growth Viable
Counts

• THE GREAT PLATE COUNT ANOMALY
• In natural samples
• Stimation of the total cells number
• Direct microscopic counts gtgtgt 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

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Direct Measurements of Microbial Growth Viable
Counts

• SOURCES OF ERRORS
• IN PLATE COUNT
• - The number of colonies depends on
• Inoculum size
• Inoculum conditions
• Selecting Culture medium
• Length of incubation Temperature of incubation
• Clumps of cell gt 1 colony gt CFU
• (colony-forming-units)

ADVANTAGES - High sensitivity - Could be made
selective
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Indirect Measurements of Microbial Growth
Turbidity

• PHOTOMETER Klett units
• - Simple filter generate light, relatively a
  narrow wavelength
• SPECTROPHOTOMETER
• 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
• ADVANTAGES
• - Quick easy
• - Do not disturb culture