Microbial Growth

1
Microbial Growth

• Chapter 6

2
Microbial Growth

• Microbial Growth Increase in the number of cells
  or in mass.
• Requirements for Growth
• Physical Factors
• Chemical Factors

3
Physical Requirements for Growth

• Temperature
• pH
• Osmotic pressure

4
Cardinal Temperatures

• The Minimum growth temperature is the lowest
  temperature at which a species will grow
• The Optimum growth temperature is the temperature
  at which a species grows best (fastest)
• The Maximum growth temperature is the highest
  temperature at which growth is possible.

5
Microbes and Temperature

• Psychrophiles (cold loving microbes )
• range 0 C - 20 C
• Mesophiles (moderate temperature loving
• microbes)
• range 20 C - 40 C
• Thermophiles (heat loving microbes)
• range 40 C - gt80 C

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pH

• Most bacteria grow between pH 6.5 and pH 7.5
• Very few can grow at below pH 4.0 (acidophilic)
• Many foods, such as sauerkraut, pickles, and
  cheeses are preserved from spoilage by acids
  produced during fermentation

7
Osmotic Pressure

• Tonicity (the ability of a solution to cause
  water movement)
• Isotonic
• Hypertonic
• Hypotonic
• In a Hypertonic solution, microbes undergo
  plasmolysis
• Water moves out of the cell,
• The cell shrinks and
• The plasma membrane separate itself from cell
  wall
• Halophiles (salt loving microbes) can tolerate
  high salt concentrations

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Chemical Requirements

• All organisms need
• 1. A carbon source.
• Chemoheterotrophs use an organic molecule, and
• Autotrophs typically use carbon dioxide.
• 2. Nitrogen is needed for protein and nucleic
  acid synthesis.
• Nitrogen can be obtained from
• Decomposition of proteins
• NH4 or NH3-
• A few bacteria such as the genus Rhizobium are
  capable of nitrogen (N2) fixation.
• 3. Water
• 4. Other chemicals required for microbial growth
  include sulfur, phosphorus, trace elements, and,
  for some microorganisms, organic growth factors

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Chemical Requirements

• On the basis of Oxygen requirements, organisms
  are classified as
• Obligate aerobes,
• Facultative anaerobes,
• Obligate anaerobes,
• Aerotolerant anaerobes,
• Microaerophiles.

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Obligate Aerobes
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Obligate Anaerobes
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Facultative Anaerobes
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Microaerophilic
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Toxic Forms of Oxygen

• Singlet oxygen (The lowest excited state of the
  dioxygen molecule) ,
• Superoxide free radical (O2-),
• Peroxide anions (O22-) and
• Hydroxyl radicals (OH)
• Free radicals Atoms or molecules with unpaired
  electrons (symbolized by a dot). Highly reactive.

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Chemical Requirements - Continued

• Aerobes, facultative anaerobes, and aerotolerant
  anaerobes must
• have enzymes against oxygen toxicity
• Superoxide dismutase (SOD)
• (2022- 2H --gt 02 H2O2)
  
• and either Catalase
• (2H202 --gt 2H20 02)
• or Peroxidase
• (H202 2H --gt 2H20).

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Culture Media

• A culture medium is any material prepared for the
  growth of bacteria in a laboratory.
• Microbes that grow and multiply in or on a
  culture medium are known as a culture.
• Agar is a common solidifying agent for a culture
  medium.

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Types of Culture Media

• Chemically Defined
• The exact chemical composition is known
• Used to grow fastidious organisms
• Complex Media
• Exact chemical composition is not known
• Most bacteria and fungi are grown with this type
  of medium.

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Special Culture Techniques

• Anaerobic Bacteria
• Reducing agents (sodium thioglycollate)
  chemically remove molecular oxygen (O2) that
  might interfere with the growth of anaerobes.
• Petri plates can be incubated in an anaerobic
  jar or anaerobic chamber (no O2, elevated CO2
  level)
• Some parasitic and fastidious bacteria must be
  cultured in living animals or in cell cultures.
• Carbon Dioxide incubators or candle jars are used
  to grow bacteria (such as microaerophilic
  bacteria) requiring an increased CO2
  concentration

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Microaerophilic Bacteria Candle Jar
lt16 O2
3 to 4 CO2
20
Microaerophilic Bacteria CO2 Generating Packet
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Selective Media

• EMB (Eosin Methylene Blue)
• Dyes (Eosin Y and Methylene blue) inhibit Gram
  () bacteria
• Selects for Gram (-) bacteria
• Eosin and Methylene blue, distinguish between
  lactose fermenting and non-lactose fermenting
  organisms.
• Lactose fermenters (left) form colonies with dark
  centers and clear borders while the non-lactose
  fermenters (right) form completely colorless
  colonies.

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Differential Media

• Differentiates between different organisms
  growing on the same plate
• Example
• Blood Agar Plates (TSA with 5 sheep blood)
• used to differentiate different types of
  Streptococci

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Alpha Hemolytic Streptococci
Incomplete lysis of RBCs
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Beta Hemolytic Streptococci
Complete lysis of RBCs
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Gamma Hemolytic Streptococci
No lysis of RBCs
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Selective and Differential Media

• Mannitol Salt Agar
• used to identify Staphylococcus aureus
• Mannitol Salt Agar
• High salt conc. (7.5) inhibits most bacteria
• sugar Mannitol
• pH Indicator (phenol red turns Yellow when
  acid)
• Staphylococcus aureus ferments mannitol and turns
  the medium yellow

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Enrichment Culture

• An enrichment culture is used to encourage the
  growth of a particular microorganism, usually in
  low concentration, from a mixed culture
• Enrichment by modifying the physical conditions
• Incubation at 55C for a thermophile
• Enrichment by modifying the nutrient content
• Adding a nutrient specific for the organism i.e.
  lactose (EMB) or a pesticide the microbe can
  dissimilate.

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Pure Culture Preservation of Cultures

• Pure Cultures
• A colony is a visible mass of microbial cells
  that theoretically arose from one cell.
• Pure cultures are usually obtained by the streak
  plate method.
• Preserving Bacterial Cultures
• Microbes can be preserved for long periods of
  time by deep-freezing or lyophilization
  (freeze-drying).

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Growth of Bacterial Cultures

• Bacterial Division
• Growth of Bacterial Cultures
• The normal reproductive method of bacteria is
  binary fission, in which a single cell divides
  into two identical cells.
• Some bacteria reproduce by budding, aerial spore
  formation (Streptomyces), or fragmentation.
• Generation Time
• The time required for a cell to divide or a
  population to double
• Logarithmic Representation of Bacterial
  Populations
• Bacterial division occurs according to a
  logarithmic (exponential) progression (2 cells, 4
  cells, 8, 16, 32, 64, 128, etc.), 2n, where n
  number of generations.

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Binary Fission (FYI)
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Phases of Growth

• 4 Phases
• Lag Phase
• Log Phase
• Stationary Phase
• Death Phase

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The Phases of Bacterial Growth

• During the lag phase, there is little or no
  change in the number of cells, but metabolic
  activity is high.
• During the log (exponential growth) phase, the
  bacteria multiply at the fastest rate possible
  under the conditions provided. The number of new
  cells formed exceeds the number of deaths
• During the stationary phase, there is an
  equilibrium between cell division and death.
• During the death phase, the number of deaths
  exceeds the number of new cells formed.

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Direct Measurementof Microbial Growth

• A standard plate count reflects the number of
  viable microbes and assumes that each bacterium
  grows into colony plate counts are reported as
  number of colony forming units (CFU).
• A plate count may be done by either the pour
  plate or the spread plate method.
• In filtration, bacteria are retained on the
  surface of a membrane filter and then transferred
  to a culture medium to grow and subsequently be
  counted.
• The most probable number (MPN) method can be used
  for microbes that will grow in a liquid medium
  it is a statistical estimation.
• In a direct microscopic count (DMC), the microbes
  in a measured volume of a bacterial suspension
  are counted with the use of a specially designed
  slide.

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Typical growth results in lactose lauryl tryptose
broth. These tubes are used to calculate an MPN
(FYI).
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MPN
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Estimating Bacterial Numbers by Indirect Methods

• A spectrophotometer is used to determine
  turbidity (optical density) by measuring the
  amount of light that passes through a suspension
  of cells.
• Plotting optical density vs. CFU/ml yields a
  roughly linear relationship and, once
  established, can be used to estimate the
  concentration of a bacterial cell suspension
  prior to performing a plate count.
• An indirect way of estimating bacterial numbers
  is measuring the metabolic activity of the
  population, for example, acid production or
  oxygen consumption.
• For filamentous organisms such as fungi,
  measuring dry weight is a convenient method of
  growth measurement.