Microbial_Growth by nuhman10


									                              BISC 115 - Human Microbiology

Lecture:   Microbial Growth

Reading: pp. 187-218


1. List the major nutritional requirements of microorganisms and describe the function
   of these essential nutrients in growth.
2. Determine each classification of a microorganism based upon: (a) the nature of its
   carbon and energy sources; (b) its requirement for oxygen; and (c) the temperature
   range in which optimal growth occurs.
3. Discuss the physical factors which may influence the growth of microorganisms.
4. Define binary fission and generation time.
5. Describe the methods used in the laboratory to measure the growth of bacteria.
   Differentiate between those direct methods that yield a viable or living cell number
   and those that provide a total cell count.
6. Draw a typical bacterial growth curve and explain the factors that contribute to the
   various phases of growth. Discuss how continuous growth at a constant rate can be
   established in a bacterial culture.

Outline of Lecture:

 I. Chemical Requirements for Growth (Nutrition)

   A. Water- solvent; major portion of cells; hydrates molecules for passage across
      cytoplasmic membrane; biochemic reactions.

   B. Carbon
      - Inorganic: Carbon dioxide; autotrophs
      - Organic: glucose; heterotrophs

   C. Nitrogen
      - Inorganic: N2 ( nitrogen gas) NO3 (Nitrate) NO2 ( Nitrites)
      - Organic: proteins, amino acids.

   D. Oxygen

       1. obligate aerobe- absolute requirement for Oxygen; about 1 atmosphere,

       2. microaerophile- require < 1atm of oxygen (about 0.2 atm)
          - many grow better with higher CO2 concentrations.

       3. obligate anaerobe
          - cannot be in presence of Oxygen at all!! It would be too toxic.
          a. aerotolerant: can tolerate some exposure to Oxygen

       4. facultative anaerobe
          - grow in presence or absence of Oxygen.

   E. Hydrogen
      - inorganic: H2S, CH4, H2 - Organic compounds: breaking down sugars, ets.
      - maintains pH, hydrogen bonds, oxidation/reduction reactions.

   F. Sulfur
      - inorganic: sulfate SO4; hydrogen sulfuid H2S
      -Organic: cysteine; methionine

   G. Phosphorus
      - inorganic phosphate (iPO4)
      - Used in:
          -Glycolysis (ATP)
          - nucleic acid synthesis
          -energy requiring reactions.

   H. Minerals
      - enzyme reactions; include calcium, iron, magnesium…..

   I.   Organic Growth Factors: vitamins, amino acids, purine and pyrimidine bases.

II. Nutritional Classification- based on nature of carbon and energy sources.

   A. Energy Source

        1. Phototroph- energy source is light

        2. Chemotroph- energy source is chemical (medically relevant)

   B. Carbon Source

        1. Autotroph- carbon source is carbon dioxide.

        2. Heterotroph- carbon source is organic

   C. Combined Terms

        1. Photoautotroph- light, CO2

        2. Photoheterotroph- light, organic

        3. Chemoautotroph- reduced inorganic compounds, CO2

        4. Chemoheterotroph- organic energy and carbon source (medically relevant)

III. Physical Factors Affecting Growth

   A. Temperature: minimum, optimum, maximum

        1. Psychrophile
           - like lower temps

       2. Mesophile (medically relevant)
          - middle temps.

       3. Thermophile
          - higher temps preferred.

   B. Hydrogen Ion Concentration (pH)
      - common optimum for pathogenic bacteria is 6-8 pH

       1. Acidophiles
          - prefer lower pH

       2. Alkaliphiles
          - prefer higher pH

   C. Radiation

       1. Visible light (photosynthesis)

       2. Ultraviolet radiation
          - inhibits bacterial growth; produce bacterial mutants.

       3. Ionizing radiation
          - X-rays, gamma rays, lethal to most microbes.

   D. Osmotic Pressure (halophiles)
      - halophiles grow in high salt concentrations in the sea.

IV. Asexual Reproduction
    - Bacteria is continuously replicating

   A. Binary Fission- one cell becomes two.

   B. Growth Rates- doubling (generation time)

V. Growth Curves

   A. Closed System or Batch Culture

       1. Lag phase
             a. Cells adjusting to new environment

       2. Log or exponential phase (best time to study bacteria)
             a. Cells actively growing and dividing at a uniform rate
             b. Determination of generation time.

       3. Stationary phase
             a. Nutrients consumed, waste products accumulate
             b. Cells dying = cells dividing
             c. Many cells surviving but unable to divide

      4. Death or decline phase
            a. Exponential dying; accumulation of toxic end products; nutrients
                depleted; autolysis release of lytic enzymes

   B. Continuous Culture (Open system)
      - add fresh medium at constant rate; remove equal amounts of culture.
      - Chemostat: maintain constant cell density by limiting essential nutrients.

VI. Laboratory Methods for Measurement of Growth

   A. Direct

      1. Petroff-Hauser counter
            a. Examine small portion of culture under microscope
            b. Count number of bacteria on calibrated slide
            c. Calculate number of bacteria per unit volume.

          a. microscope
                 a. problem is that we cant distinguish live from dead cells.
          b. total cell count

      2. Coulter counter
            a. As cells pass through device, trigger or electric sensor that tallies their

          a. electronic
          b. total cell count and distribution of sizes

      3. Colony formation on agar plates

          a. dilution methods
                   a. serial dilution of culture
                   b. In solution, look for colonies if dead no colonies will form
                             i. Count number of colony forming unit (cfu)
                            ii. Use plates with countable fractier (30-300 cfu)
                           iii. Multiply number of colonies by dilution factor.
          b. filtration (used for dilute culture)
                   a. filter through membrane
                   b. place membrane on agar plate; count colonies

   B. Indirect

      1. Cell density
         - use spectrophotometer to measure turbidity (how cloudy sample is)

          a. turbidity or optical density
          b. spectrophotometer
             - indirect, must generate standardized curve for future reference

      3. Cell mass
             a. Measure total wet or dry weight

       b. Measure total amount of some cellular component (protein, RNA, etc.)

4. Metabolic activity
      a. Measure enzyme activity
              i. Products (oxyten, ATP, etc.)

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