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Measurement of Growth Contd'

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Phase contrast microscope required, when cells not stained. Not suitable for cell ... when pH raised acidophilic bacterial membranes destroyed and cells lyse ... – PowerPoint PPT presentation

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Title: Measurement of Growth Contd'


1
Measurement of Growth (Contd.)
  • Direct Measurement
  • Total cell count using a microscope (phase
    contrast)
  • Samples dried on a slide
  • Liquid samples counting chambers

2
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3
Limitations of Microscopic counts (Total cell
count)
  • Dead cells cannot be distinguished from living
    cells
  • Small cells maybe missed
  • Precision is hard to achieve
  • Phase contrast microscope required, when cells
    not stained
  • Not suitable for cell suspensions of low density
  • Motile cells have to be immobilized before
    counting

4
Viable cell count
  • Count only living cells
  • Assumption Each viable cell can grow and divide
    to yield one colony
  • Plate count /Colony count
  • Viable counts
  • 1. Spread plate method
  • 2. Pour plate method

5
Viable cell counts (Contd.)
6
30-300 colonies statistically significant
7
Sources of Error in Plate Counts
  • Results depend on inoculum size, suitability of
    medium and incubation conditions
  • Small colonies maybe missed during counting
  • Incorrect procedure (pipetting, dilution samples
    etc.)
  • Results in cfus as more than one cell maybe
    present in a cfu
  • Food product assessment (highly sensitive)
  • Unsuitable for natural samples such as soil and
    water..Why ?

8
Indirect measure of growth Turbidity
Turbid cloudy Optical density units
(OD) Standard curve has to be generated relating
a direct measure (plate count) to OD obtained
9
Indirect measure of growth Turbidity (Contd.)
Calculate generation time Estimate of colonies
from plating a culture of a given OD Relationship
between OD and colonies invalid at high turbidity

10
Chemostat Continuous Culture
  • Continuous culture is an open system unlike a
    batch culture
  • Maintains cells in exponential phase for longer
    periods of time
  • Adding fresh culture and removing used culture at
    a constant rate chemostat
  • Needed for studies which look at exponential
    phase cells ex. synthesis of an enzyme

11
Chemostat
12
Chemostat (Contd.)
  • In a chemostat, the rate at which the culture is
    diluted governs the growth rate and growth yield
  • Unlike a batch culture, growth rate and yield
    controlled independently
  • Growth rate adjusting dilution rate
  • Yield varying concentration of a nutrient

13
Chemostat (Contd.)
  • The population size is governed by the
    concentration of the growth-limiting nutrient
    entering the vessel
  • Growth rate (doubling time) varies, while
    population density remains constant
  • Washout when dilution too high
  • Cell Death when dilution too low

14
Environmental factors that affect microbial
growth
  • Temperature
  • pH
  • Osmotic effects
  • Oxygen

15
Temperature
  • Microorganisms have different temperature
    requirements for optimal growth
  • Temperature can have one of two effects
  • Temperature rises-enzymatic reactions in the cell
    speed up and growth is accelerated
  • Temperature rises exceedingly- denaturation of
    proteins
  • Cardinal Temperature
  • Minimum temperature-below which growth will not
    occur
  • Optimum temperature- growth is most rapid
  • Maximum temperature- above which growth is not
    possible
  • Temperature optimum can be altered by other
    factors ex. composition of the growth medium

16
Cardinal Temperature
Ex. Cardinal Temperatures for E.coli optimum
39 C, maximum 48 C minimum 8 C (typical range
for any organism is between 30-40 degrees)
17
Temperature Classes
Thermophile Thermus aquaticus, Taq polymerase
18
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19
Effect of pH on growth
  • Organisms that grow at low pH acidophiles
  • Fungi more acidophilic than bacteria
  • Stability of cytoplasmic membrane when pH raised
    acidophilic bacterial membranes destroyed and
    cells lyse
  • Indicative of high concentration of hydrogen ions
    required for membrane stability
  • Organisms that require high pH Alkaliphiles
  • Area of research interest Can a PMF be set up in
    such an alkaline membrane?
  • Na gradient
  • PMF

20
Internal pH maintained at neutral Buffers used
in culturing
21
Osmotic effects on growth
  • Water is the primary biological solvent
  • Water availability expressed in physical term as
    water activity (aw)
  • aw is the ratio of vapor pressure of air in
    equilibrium with a substance or solution to the
    vapor pressure of pure water
  • aw 1.00 for pure water and aw 0.98 for sea
    water and aw is 0.75 for salt lakes
  • Every organism has an optimum and a range of
    tolerance.
  • Most cells are unable to cope with environments
    of lowered water activity (higher solute
    concentrations) and either die or become
    dehydrated or dormant

22
Osmotic effects on growth (Contd.)
  • Water diffuses from region of high water
    concentration (low solute) to a region of low
    water concentration (higher solute
    concentration) Osmosis
  • Water flows into cell positive water balance
  • Halophiles salt
  • Osmophiles sugar
  • Xerophiles dry

23
How do cells grow under conditions of low water
activity?
  • Increasing internal solute concentration
  • Pumping inorganic ions into cell from the
    environment
  • Synthesizing or concentrating an organic solute
  • Solute should be noninhibitory to other chemical
    processes within the cell compatible solutes

24
Effect of Oxygen on growth
  • Aerobes require oxygen
  • Facultative oxic or anoxic
  • Anaerobes
  • Aerotolerant anaerobes
  • Obligate anaerobes Clostridium

25
Thioglycolate medium
Anoxic Jar
26
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27
Catalase Test rids cells of hydrogen peroxide
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