CHAPTER 6 - Microbial Growth

Note: These notes are outlines of my lectures and they in no way represent all the material covered in class!!

Physical requirements for the growth of microorganisms

WHAT FACTORS AFFECT GROWTH?

I. PHYSICAL FACTORS:

1. Temperature:
How does temperature affect optimal growth?

2. pH: usually narrow range of growth most don't grow below pH 4 and above pH 9 internal pH always near neutrality may produce end products that change local pH/need buffers acidophiles - pH 1 - 5.5
  • neutrophiles - pH 5.5 - pH 8.5
  • alkalophiles - pH 8.5 - 11.5

    •  
    Bacteria min pH opt pH max pH
    Thiobacillus 1.0 2-2.8 4-6
    E. coli 4.4 6-7 9.0
    C. sporogenes 5.4 6-7.6 9.0
    P. aeruginosa 5.6 6.6-7 8.0
    Nitrobacter 6.6 6.6-8.6 10.0

    3. Classification based on water activity/osmotic pressure (remember, low water activity = high osmotic pressure)

    How do microbes adapt to low water activity?


    4. Oxygen:

    How does oxygen affect optimal growth?


    Why is oxygen toxic to some microbes?

    Organic growth factors

    commonly called vitamins, these are organic substances required for the growth of an organism but which the organism can not synthesize.

    What is the difference between a defined and an undefined medium?

    WHAT FACTORS AFFECT GROWTH?


    I.  Nutrients:
     
    ELEMENT   CELL FUNCTION
    C   backbone of organic cell components, energy
    H   water, organic components, pH, hydrogen bonds, re-dox
    O   water, organic components, respiration
    N   amino acids, nucleotides, coenzymes, ATP
    S   amino acids, coenzymes, enzymes
    P   nucleic acids, phospholipids, coenzymes, ATP
    Fe   cytochromes, enzymes
    Na, K, Ca, Cl, Mg, Mn   Trace elements: transport, ionic balance, cofactors (e- donor/acceptors)

    II. CLASSIFICATION OF ORGANISMS BASED ON carbon, energy, and electron sources

    Bacterial Growth

    Mathematics of growth- generation time

    Growth equation:

        n = log10Nf - log10 Ni
                     0.301

                                            Where n = number of generations, Nf = final conc. of cell (e.g. 109/ml),
                                                        Ni =  initial  conc. of cells (e.g. 103/ml), and 0.301 is factor to convert log2 to log10.  Example: measure culture at 9 a.m.: No = 10,000 cells/ml.  Then,  measure culture at 3 p.m.: Nf = 100,000
        cells/ml. Calculate n = (5 - 4)/0.3 = 1/0.3 = 3.33 generations.

    Generation time: Total time = 6 hours = 360 minutes/3.33 generations = 108 minutes/generation
                                                Conclude: generation time = 108 minutes

    Note: be able to calculate g.t. Pay attention to units!

    Graphical measurement of growth
    · Plotting # of cells vs. time gives a curved line.
    · Plotting log # of cells vs. time gives a straight line --- easier to interpolate, use.
    · Plotting # of cells vs time on semilog paper also gives a straight line --- easiest way in
        practice to work with growth measurements.
    · Note: often what is plotted on the Y-axis of semilog paper is not # of cells, but something
        more easily measurable, such as Absorbance (see below).
    .

    CULTURE MEDIA
    A culture medium is any material prepared for growth of an organism in a laboratory setting. Microbes that can be cultured on a petri-plate or in a test-tube containing media are said to grow under in vitro conditions ("within-glass".)

    It was not until the era of Robert Koch and his coworkers that Agar was introduced as a a common medium for bacterial growth. Agar is a complex polysaccharide derived from a marine sea weed. Few bacteria possess enzymes capable of digesting agar and therefore it is useful as a solidifying agent and for isolating microbes in pure culture. Prior to the advent of agar, gelatin was used as a growth medium. Unfortunately, many bacteria possess enzymes that liquify gelatin and therefore this medium is not useful for isolating pure cultures. However, gelatin liquefaction is one among a series of biochemical tests that helps differentiate species of bacteria.

    What is a PURE CULTURE?

    Media vary in their chemical composition. In turn, the composition of the media determines microbial growth and the type of microbes that will grow.

    a) Chemically defined media: exact chemical composition is known. Such media is often
        commercially prepared.

    b) Selective media. Contain chemicals which encourage growth of certain types of microbes
        but inhibits the growth of others.

    c) Differential media allows different microbes to be distinguished on the basis of various
        biochemical reactions. Fermentation reactions involving the catabolism of various sugars are
        particularly useful biochemical tests


    d) Enrichment media contains a rich supply of nutrients to encourage the encourage
        growth of microorganisms. A commonly used enrichment medium is blood agar. This
        medium is also differential and it permits detection of differnt patterns of hemolysis.

     Measurement of growth

    a) Total Cell count
    · Petroff-Hausser chamber slide -- needs large conc. (107 cells/ml minimum).
    · Coulter Counter (for larger microbes; fungi, yeasts, protozoa, etc.) --- uses electrical charge
        difference in passing through small hole. Not so useful with bacteria, get errors due to
        clumping, debris, unable to differentiate bewteen live and dead cells, etc.

    b) Viable count
    CFU (colony forming units) assay
    1. carry out dilution series
    2. plate known volumes on plates
    3. count only plates with 30 - 300 colonies (best statistical accuracy)
    4. extrapolate to undiluted cell conc.

    Measures colony forming units (CFU), may or may not be same as number of cells --- accurate, but requires time for incubation.

    Two ways to carry out viable count:
        1. Spread plate: bacteria are spread on the surface of agar using some sterile spreading
            device. Advantages: if properly carried out, all colonies should be easily counted.
            Disadvantages: takes some time, not always reliable in inexperienced hands, cells with low
            tolerance to oxygen will not grow. If "spreaders" are present may overgrow plate surface.

        2. Pour plate: bacteria are mixed with melted agar and cooled; colonies grow throughout the
            agar. Advantages: almost fail-proof technique, colonies well separated. Can allow growth
            of organisms with lower oxygen tolerance in agar. Disadvantages: colonies variable size,
            harder to see similarity in colony morphology between those on surface and in agar.
            Counting may be more difficult. Heat may kill some cells before agar cools and gels.

    c) Light techniques
    Often, can estimate cell numbers accurately by measuring visible turbidity. Light scattered is proportional to number of cells. This only works above cell densities of 107 in pure cultures. Eyeball method. This is not a precise measurement, but shoud allow estimation within an order of magnitude.

    • no turbidity means less than 107 cells/ml
    • Slight turbidity = 107 - 108 cells/ml
    • high turbidity= 108 - 109 cells/ml
    • Very. high turbidity = greater than 109 cells/ml (cultures rarely get as high as 1010 cells/ml)

    Absorbance (usually at wavelengths around 400-600 mn). Accurate measure of cells when concentration not too high. Easy and quick to measure (can sample in less than a minute).

    d) Batch vs. Continuous culture methods
    · Batch method: put small inoculum of pure culture into sterile medium, let grow. Common lab
        procedure, but not typical of many real environments.
    • Continuous culture (see text section 6.11): use chemostat or turbidostat. Trickle fresh
        medium into culture at slow but steady rate, displace = volume of culture as overflow.
    • Cells remain in exponential (but suboptimal) state, growing at known rate. Good simulation for
        study of many natural environments.