Growth of Bacteria

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Growth of Bacteria
How do bacteria grow?
Measured as increase in cell number not size.
Transverse binary fission or Binary Fission
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Goals for Chapter
How do we measure cell growth?
What are the stages of cell growth?
What is happening in cells during cell growth?
What are the effects of environmental conditions
on cell growth?
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Cell division
What is happening?
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What can we measure to determine if growth is
occurring?
Cell number
Direct methods
Indirect methods
Microscopy
Culture density
Dry cell weight
Viable cell count
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Microscopy measures dead and live cells
Petroff- Hauser counter
Direct method
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Measure Colony Forming Units
CFUs
Viable cell counts
Does each colony represent one cell?
Cell must be alive and able to grow
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Count between 30 and 300 colonies
Dilution plating Why is this necessary?
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Measuring cell turbidity
Spectrophotometer
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Describing growth in a closed system
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What is happening at each stage?
Cells are adapting to environment, enzymes
synthesized DNA replicated All cells are not
physiologically the same
Lag phase
All cells growing at same rate Cells have on
average the same types and number of
macromolecules
Log phase or Exponential phase
Cells are not all dividing at the same time!
Cells are dividing and dying at the same rate
Stationary phase
More cells are dying than dividing
Decline phase
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How do we calculate growth rate?
During exponential phase, growth is by geometric
progression 1, 2, 4, 8, etc. or 20, 21, 22,
23.........2n (where n the number of
generations)
k Growth rate constant generations/timen/t
g generation time or doubling time t/n
t time n number of generations
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Generation times for some common bacteria under
optimal conditions of growth.
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Chemostat- used to study mixed cultures or
physiological properties of cells
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What kinds of questions can we ask using growth
experiments?
What is the effect of temperature on growth?
What is the effect of pH on growth?
What is the effect of Oxygen on growth?
What is the effect of salt on growth?
What is the effect of ______________ on growth?
A growth experiment
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Effect of temperature on growth
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Temperature
Figure 6.1
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Psychrotrophs
Figure 6.2
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The Requirements for Growth Physical Requirements

• pH
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• Acidophiles grow in acidic environments

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The Requirements for Growth Physical Requirements

• Osmotic Pressure
• Hypertonic environments, increase salt or sugar,
  cause plasmolysis
• Extreme or obligate halophiles require high
  osmotic pressure
• Facultative halophiles tolerate high osmotic
  pressure

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The Requirements for Growth Physical Requirements
Figure 6.4
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The Requirements for Growth Chemical Requirements

• Oxygen (O2)

obligate aerobes Faultative anaerobes Obligate anaerobes Aerotolerant anaerobes Microaerophiles

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What are the effects of Oxygen on growth?
Many forms of life require oxygen for
_________________.
aerobic respiration
Some forms of oxygen can actually be toxic!
Metabolism, UV light, chemical reactions can
create toxic forms of oxygen
Aerobes have enzymes to detoxify toxic forms of
oxygen.
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Toxic Forms of Oxygen

• Singlet oxygen O2 boosted to a higher-energy
  state
• Superoxide free radicals O2?
• Peroxide anion O22?
• Hydroxyl radical (OH?)

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Superoxide Dismutase

• Catalyzes the following reaction in which
  superoxide free radicals are converted into
  molecular oxygen and hydrogen peroxide
• O2- O2- 2 H H2O2
  O2(g)

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Catalase

• Catalyzes the conversion of hydrogen peroxide to
  water and oxygen.
• 2 H2O2 2 H2O O2(g)

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Catalase Activity

• Negative Positive
• All photographic material taken from
  Bacteria ID - A Computer Simulation of Bacterial
  Identification (compact disc), by Konopka,
  Furbacher and Gedney.

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Peroxidase

• Also catalyzes the breakdown of hydrogen peroxide
• Differs from catalase - does not produce oxygen
• H2O2 2 H 2 H2O
• ex. Streptococcus

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The Requirements for Growth Chemical Requirements

• Carbon
• Structural organic molecules, energy source
• Chemoheterotrophs use organic carbon sources
• Autotrophs use CO2

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The Requirements for Growth Chemical Requirements

• Nitrogen
• In amino acids, proteins
• Most bacteria decompose proteins
• Some bacteria use NH4 or NO3?
• A few bacteria use N2 in nitrogen fixation
• Sulfur
• In amino acids, thiamine, biotin
• Most bacteria decompose proteins
• Some bacteria use SO42? or H2S
• Phosphorus
• In DNA, RNA, ATP, and membranes
• PO43? is a source of phosphorus

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The Requirements for Growth Chemical Requirements

• Trace Elements
• Inorganic elements required in small amounts
• Usually as enzyme cofactors

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The Requirements for Growth Chemical Requirements

• Organic Growth Factors
• Organic compounds obtained from the environment
• Vitamins, amino acids, purines, pyrimidines

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Culture Media

• Culture Medium Nutrients prepared for microbial
  growth
• Sterile No living microbes
• Inoculum Introduction of microbes into medium
• Culture Microbes growing in/on culture medium

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Agar

• Complex polysaccharide
• Used as solidifying agent for culture media in
  Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100C
• Solidifies 40C

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Culture Media

• Chemically Defined Media Exact chemical
  composition is known
• Complex Media Extracts and digests of yeasts,
  meat, or plants
• Nutrient broth
• Nutrient agar

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Culture Media
Table 6.2 6.4
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Anaerobic Culture Methods

• Reducing media
• Contain chemicals (thioglycollate or oxyrase)
  that combine O2
• Heated to drive off O2

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Anaerobic Culture Methods

• Anaerobic jar

Figure 6.5
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Anaerobic Culture Methods

• Anaerobic chamber

Figure 6.6
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Capnophiles require high CO2

• Candle jar
• CO2-packet

Figure 6.7
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Selective Media

• Suppress unwanted microbes and encourage desired
  microbes.

Figure 6.9b, c
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Differential Media

• Make it easy to distinguish colonies of different
  microbes.

Figure 6.9a
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Enrichment Media

• Encourages growth of desired microbe
• Assume a soil sample contains a few
  phenol-degrading bacteria and thousands of other
  bacteria
• Inoculate phenol-containing culture medium with
  the soil and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Transfer 1 ml to another flask of the phenol
  medium and incubate
• Only phenol-metabolizing bacteria will be growing

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• A pure culture contains only one species or
  strain
• A colony is a population of cells arising from a
  single cell or spore or from a group of attached
  cells
• A colony is often called a colony-forming unit
  (CFU)

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Streak Plate
Figure 6.10a, b