Measurement of Growth Contd'

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Measurement of Growth (Contd.)

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

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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

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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

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Viable cell counts (Contd.)
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30-300 colonies statistically significant
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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 ?

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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
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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

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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

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Chemostat
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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

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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

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Environmental factors that affect microbial
growth

• Temperature
• pH
• Osmotic effects
• Oxygen

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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

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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)
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Temperature Classes
Thermophile Thermus aquaticus, Taq polymerase
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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

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Internal pH maintained at neutral Buffers used
in culturing
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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

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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

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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

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Effect of Oxygen on growth

• Aerobes require oxygen
• Facultative oxic or anoxic
• Anaerobes
• Aerotolerant anaerobes
• Obligate anaerobes Clostridium

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Thioglycolate medium
Anoxic Jar
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Catalase Test rids cells of hydrogen peroxide