Chapter4 Microbial growth

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Chapter4 Microbial growth

• Isolation of pure cultures
• the growth curve
• Measurement of microbial growth
• The continuous culture of microorganisms

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Isolation of pure cultures

• Pure culture a population of cells arising from
  a single cell, to characterize an individual
  species. They are required for the careful study
  of an individual microbial species.
• Single colony
• How to get
• 1. Dilute-pour/spread plate technique
• dilute?pour/spread ?isolated colonies
• 2. Streak plates inoculating loop

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Petri Disk Cultivation of a Pure Strain
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The Streak Plate Technique to obtain a pure
clone
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Laboratory Culture of Microorganisms
1.5 Agar
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Colony morphology and growth

• individual species often form colonies
  of characteristic size and appearance.

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The growth of colonies on agar

• At the colony edge cells grow at maximum rates
• In the center of colony cells are lying growth
  is much slower

Cause Oxygen, nutrients and toxic products
The colony center is much thicker than the
edge. Cell-cell communication and quorum sensing
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Microbial growth

• Batch culture  When microorganisms are grown in
  a closed system, population growth remains
  exponential for only a few generations and then
  enters a stationary phase due to factors like
  nutrient limitation and waste accumulation.
• Continuous Culture If a population is cultured
  in an open system with continual nutrient
  addition and waste removal, the exponential phase
  can be maintained for long periods.

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The growth curve The logarithm of the
number of viable cells versus the incubation
time.
.
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The Growth Cycle of Populations

• (a) Lag phase cells begin to synthesize
  inducible enzymes and use stored food reserves.
• (b) Logarithmic growth phase the rate of
  multiplication is constant.
• (c) Stationary phase death rate is equal to rate
  of increase.
• (d) Death phase cells begin to die at a more
  rapid rate than that of reproduction.

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Lag phase

• no immediate increase in cell mumber. The cell is
  synthesizing new compoments.
• ATP, cofactor, ribosomes, enzymes.
• The lag phase varies in length with the condition
  of the microorganisms and the nature of the
  medium.
• How to shorten the lag phase?

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Exponential phase/log phase

• Microorganisms are growing and dividing at the
  maximal rate possible.
• The population is most uniform in terms of
  chemical and physiological properties.
• Such culture biochemical and physiological
  studies.

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Stationary phase

• Balance between cell division and cell death.
• Poplation growth ceases and the growth curve
  becomes horizontal.
• Bacteria 109 cells per ml
• Protozoan and algae 106 cells per ml.
• Reasons 1. Nutrient limitation
• 2. The accumulation of toxic waste
  products
• 3. A critical population level is
  reached

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Death phase

• The decline in the number of viable cells.
• Starvation can be a positive experience for
  bacteria.
• 1. decrease overall size protoplast
  shrinkage
• 
  nucleoid condensation.
• 2. Produce a variety of starvation proteins.
• 3. Increase peptidoglycan cross-linking and
  cell wall strength.
• 4. The Dps protein protects DNA.
• 5. Chaperones prevent protein denaturation
  and renature damaged proteins.

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Cell life cycle in Eukaryotic cells
G1 Primary growth phase of the cell during
which cell enlargement occurs, a gap phase
separating cell growth from replication of the
genome
S phase in which replication of the genome
occurs
G2 Phase in which the cell prepares for
separation of the replicated genomes, this phase
includes synthesis of microtubules and
condensation of DNA to form coherent chromosomes,
a gap phase separating chromosome replication
from miosis.
M phase called miosis during which the
microtubular apparatus is associated and
subsequently used to pull apart the sister
chromosomes.
Eukaryotic cell Prokaryotic cell
G1 S G2 M G1
R D
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generation time
The time required for a cell to divide (and its
population to double) is called the generation
time.
Suppose that a bacterial population increases
from103 cells to 109 cells in 10 hours. Calculate
the generation time.
Nt No x 2n
G t log2 / log Nt log No
No number of bacteria at beginning of time
interval. Nt number of bacteria at end of any
interval of time (t). G generation time T
time , usually expressed in minutes n number of
generation
Number of cells
Time
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Generation time

• Example
• 100 bacteria present at time 0
• If generation time is 2 hr
• After 8 hr mass 100 x 24

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3 types of cell divisiona. Binary Fission b.
Buddingc. Multiple Fission
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Binary fision
Most bacterial cells reproduce asexually by
binary fision, a process in which a cell divides
to produce two nearly equal-sized progeny cells.
Binary fision involves three processes
Increase in cell size (cell elongation), DNA
replication Cell division
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Binary Fission (???)
Attention difference between cocci and bacilli
in binary fission
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Budding (??)
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Multiple Fission
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Measurement of microbial growth

• Population mass
• Population number
• No single technique is always best.

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Measurement of cell numbers

• Total cell count (direct observation under
  microscope)
• Viable count (plate count or colony count)
• Spread plate method
• Pour plate method
• The membrane filtration procedure
• The plates required to have between 30 and 300
  colonies.

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Direct counting

• The counting chamber

Disadvantages 1. The microbial population must
be fairly large for accuracy because such a small
volume is sampled. 2. Difficult to distinguish
between living and dead cells

• Electronic counters the Coulter Counter
• for larger microorganisms such as protozoa,
  algae, and nonfilamentous yeasts.

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Viable count Spread plate methodand Pour plate
method
Problems 1. Clumps of cells. colony forming
units(CFU) 2. employed agar medium cannot
support growth of all the viable microorganisms
present. 3. Hot agar spread platesgtpour plates

• Three basic steps
• Dilution,
• Plating
• Incubation

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Measuring viable bacteria
Colony forming units
colony
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Viable Count Dilution
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Viable Count Plating
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Incubation (and counting)
Pour plate method
Spread plate method
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The membrane filtration procedure

• Analyze aquatic samples

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Measurement of cell mass

• 1. Dry weight useful for measuring the growth of
  fungi.
• cells growing in liquid medium are collected by
  centrifugation, washed, dried in an oven, and
  weighed.
• for bacteria, not very sensitive
• 2. Turbidity and microbial mass measurement
• 3. The amount of a substance
• total protein or nitrogen
• chlorophyII(algae)

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Measuring bacterial mass (live dead) in liquid
culture
Turbidity (Cloudiness)
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Measuring cell density Turbidity vs Cell density
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The continuous culture of microorganisms

• Continual provision of nutrients and removal of
  wastes.
• A microbial population can be maintained in the
  exponential growth phase and at a constant
  biomass concentration for extended periods in a
  continuous culture system.

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• The chemostat
• Chemostat used for continuous cultures,
  rate of growth can be controlled either by
  controlling the rate at which new medium enters
  the growth chamber or by limiting a required
  growth factor in the medium
• The turbidostat
• measures the absorbance or turbidity of the
  culture in the growth vessel.
• automatically regulated to maintain a
  predetermined turbidity or cell density.

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How to maintain Exponential Growth?
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Questions

• How to obtain a pure culture? How to calculate
  cell growth?
• How to describe the colony morphology?
• How cells divide themselves? How to do viable
  count?
• What is the microbial cell growth cycle?
• How to measure the cell mass and number?
• What is continuous culture, chemostat,
  turbidostat?
• How to maintain exponential growth?