Chapter 8

• Bacterial Growth in the Lab

  • May be grown on agar plates containing nutrients
  • May also be grown in brother either aerated (shaking) or not
  • Growth in nutrient rich media takes on form of logarithmic growth
  • Binary fission: 1, 2, 4, 8, 16
  • Doubling time: growth rate
  • E coli has doubling time in rich broth culture of 20 minutes
• Measuring Microbial Growth

  • All of these measurements are performed on liquid cultures only
  • Plate counts: count number of colony forming units (CFU) on plate
  • Optical Density (turbidity): directly measure the ability to absorb light at a specific wavelength
  • Microscopic visualization: count bacteria in a given amount of culture directly
• Plate Counts

  • Dilute culture in appropriate buffer/medium
  • Plate onto nutrient agar plates
  • Count CFUs after right amount of time (24h for standard)
  • Advantage: only count bacteria capable of dividing
  • Disadvantage: takes time to do so, subject to dilution error
• Optical Density (Turbidity)

  • Use spectrophotometer
  • Shines light at specific wavelength through culture, measures amount that passes through (transmittance or its converse, absorbance)
  • AdvantageL quick, accurate method for determining density of culture
  • Disadvantage: may count dead cells, has narrow range of effectiveness
• Microscopy

  • Take direct sample, count cells: use Hemocytometer or Petroff-Hauser cell count
  • Advantage: quick, precise
  • Disadvantage: vary from field to field, so need to observe many fields to get statistically relevant numbers, doesn't distinguish dead from live cells, intensive compared to other two techniques
• Logarithmic Growth

  • Bacterial culture growth has 4 phases
  • Lag: inoculating a culture, cells take time to adjust to new environments
  • Log phase: maximal growth rate
  • Stationary phase: number of cells is steady
  • Death phase: rate of cell death exceeds division rate
• Batch Versus Continuous Culture

  • The growth curve is for batch cultures: microbes are exposed to the same media, even as they utilize the nutrients and release various products
  • Continuous culture is done in a chemostat: spent media is removed, and fresh media is added, at a constant flow rate: used in industrial fermentations
• Chemostat Cultures

  • At low dilution rates, growth rate proportional to D
  • Maintenance energy: energy used to keep bacteria at homeostasis
  • High dilution rates: washout
• Environmental Factors

  • Growth rates affected by many factors: temperature, pH, water activity, oxygen
  • Optimal growth rate will reflect niches: deep sea vents, high pressure, temperature, anoxic, extremophiles
• Temperature

  • Psychrophilic: cold loving
  • Mesophile: room temperature
  • Thermophile: heat loving
  • Hyperthermophile: extreme thermophile
• Cardinal Temperatures

  • Listeria monocytogenes: 30-37
  • Vibrio marinus: 15
  • Pseudomonas maltophilia: 35
  • Thiobacillus novellus: 25-30
  • Staphylococcus aureus: 30-37
  • Escherichia coli: 37
• pH Optimum

  • Acidophile: 1-4.5
  • Neutrophile: 5.5-8.5
  • Alkalophile: 7.5-11.5
  • Note doesn't generally extend to max pH 14
• Solute Concentration

  • Hypotonic solution: membrane stretches, swelling, turgor pressure, normal situations for many organisms
  • Hypertonic solution: membrane shrinks from cell wall, plasmolysis, compatible solutes
• Water Activity

  • Osmotic pressure: solutes (salts: halophile)
  • Matric pressure: adsoprtion to solids (desiccation)
  • Water activity: available water for organisms
  • Low water activity: makes life difficult
• Other Factors on Microbial Growth

  • Radiation: UV ionizing, DNA damage T-T dimers, Deinococcus radiodurans, endospores
  • Pressure: barophilic (piezophilic, deep sea), barotolerant
• Environmental Factors

  • Leibig's law of the minimum: total biomass of organism determined by nutrient present at lowest concentration
  • Shelford's law of tolerance: above or below certain environmental limits, a microorganism will not grow, regardless of nutrient supply