Bacterial Growth

• Bacterial Growth


  Methods to Determine Bacterial Growth
• Total counts methods

  • Direct microscopic counting using Helber or haemocytometer counting chambers
  • Coulter counter
  • Turbidity (how cloudy something is/opacity) methods
  • Dry weight determinations
  • Nitrogen, protein or nucleic acid determinations
• Total Counts


  Gives a total number of bacteria and doesn't distinguish between dead and alive bacteria
• Coulter counter


  Directly counts cells as they interrupt an electrical current flowing across a narrow tube in front of an electronic detector
• Coulter counter can determine the size of the specimen you wish to count, however, debris in media and presence of clumps can give inaccurate results
• Viable Counts


  Methods that can distinguish between dead and alive bacteria
• Viable Count Methods


  • Grow until we can see a colony by eye
  • Pour plate — counting colonies in agar
  • Surface spread/surface drop (Miles Misra) — counting colonies on agar surface
  • Membrane filter methods — colonies growing on membranes on agar surface
  • MPN (most probable number) — counts based on the proportion of liquid cultures growing after receiving low inocula
• Rapid Methods (indirect viable counts)


  • Epifluorescence — uses dyes that give characteristic fluorescence only in living cell and often coupled to image analysis
  • ATP methods — measure ATP production in living cells using bioluminescence
  • Impedance — measures changes in resistance, capacitance or impedance in growing cultures
  • Manometric methods — measure oxygen consumption or carbon dioxide production by growing cultures
• Determination of cell constituents

  Measure specific cell material such as DNA
• Colony Forming Unit (CFU)


  A measure of viable (capable of living successfully) cells in which a colony represents an aggregate (collection) of cells derived from a single progenitor cell or a small cluster of them
• 1 bacteria is potentially a CFU. 1 bacteria/CFU is invisible to the naked eye, so we have to use a microscope it see it. A CFU is capable of forming one colony
• As bacteria grow and divide by making clones of themselves, the colony becomes visible to the naked eye
• What can CFU be used to determine?

  Used to determine the number of viable bacterial cells in a sample/CFU per 'ml'. This will relate to the degree of contamination in the sample or the magnitude of an infection
• Bacterial Growth Curve


  1. Lag phase

  2. Log (exponential) phase

  3. Stationary phase

  4. Death phase
• Lag phase


  The bacteria are getting used to the nutrients/environment; there's no dividing of bacteria
• Log (exponential) phase


  Bacteria divide exponentially as 1 will divide into 2, 2 will divide in 4 etc, which is exponential growth
• Stationary phase


  The rate of growth = rate of death: there are some cells that have stopped dividing, some that are actively dividing and some that are dying, as the nutrients are starting to be used up
• There are bacteria that respire, which produce carbon dioxide, making the environment acidic. The more carbon dioxide there is, the more acidic the environment
• Death phase


  Bacteria die as they run out of nutrients
• Bacterial types by temperature range

  • Psychrophile 20°C 40°C to 20°C
  • Mesophile 20°C to 40°C bacteria that are pathogenic and live on or in our bodies
  • Thermophile 40°C 40°C to 85°C
• Permissive temperatures


  The range of temperatures that bacteria can actively grow and multiply in
• For every organism, there's a minimum temperature below which no growth happens, an optimum temp at which growth is the fastest and a max temp above which growth is not possible
• At 37°C, pathogens multiply most quicky, but as temperature increases/decreases, the rate slows down. At low temps, bacteria become dormant; they aren't killed off. At high temperatures (above their optimum temperature), bacteria can be killed
• As temperatures rise, chemical and enzyme reactions within the cell proceed more rapidly and growth becomes faster until an optimal rate is reached. However, beyond this temp, certain proteins become denatured due to thermal lysis, resulting in a rapid loss of cell viability, i.e. cells rapidly begin to die
• Bacterial Nutrient Requirements


  • Energy source
  • Carbon source
  • Nitrogen source
  • Potassium
  • Trace elements
• Chemolithotrophs can derive much of their nutrition from simple inorganic forms of these elements, including using atmospheric carbon dioxide and nitrogen as sources
• Majority of bacteria require a fixed carbon source, usually in the form of a sugar, but may also be obtained from complex organic molecules such as benzene, paraffin waxes and proteins
• Bacteria generally obtain nitrogen from ammonium ions but can also get it via the deamination amino acids, which can provide sources of C and N simultaneously
• Many classes of bacteria are auxotrophic and can grow on simple sugars together with ammonium ions, a source of potassium and trace elements. They can synthesise all the amino acids and ancillary (provides necessary support for its activities) factors required for growth and division
• The nature of the C and N sources determines the rate of growth: a faster rate of growth is often obtained when glucose or succinate is the C source rather than lactose or glycerol and when amino acids are the source of N rather than ammonium salts
• If faced with a choice of C and N sources, the bacteria will adapt their physiology to the preferred substrate and only when this is depleted will they turn their attention to the less preferred substrate
• All eukaryotes are photoautotrophic (capable of using light as the energy source in the synthesis of food from inorganic matter) or heterotrophic (organic compounds are used for energy and C sources and there's a limit to what can be used for energy and C sources, i.e. getting food from other plants or animals, or relating to such living things)
• Bacteria can use combination of inorganic compounds such as ammonia or hydrogen, or energy sources such as light as well as a combination of carbon dioxide or organic compounds as their C source
• This allows them to survive in a range of different environments
• Growth Factors


  • Purines and pyrimidines - required for synthesis of nucleic acids DNA and RNA
  • Amino acids - required for protein synthesis
  • Vitamins - needed as coenzymes and functional groups of certain enzymes
• Some bacteria don't need growth factors
• Auxotrophs


  Bacterial strains that need growth factors not needed by wild-type strains
• Water activity (Aᵥᵥ)


  The vapour pressure of water in the space above the material relative to the vapour pressure above pure water at the same temperature and pressure
• Aᵥᵥ of pure water (has no solutes) is 1.0. The more solutes, the lower the water activity
• Bacterial types by water activity

  • Halophiles - need NaCl for growth: Mild 1.6%, Moderate 6-15%, Extreme 15-30%
  • Osmophiles - survive in high sugar environments
  • Xerophiles - live in dry environments, but still need a little bit of water to survive