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Microbial Growth

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Last revised: Wednesday, February 9, 2000
Ch. 6 in Prescott et al, Microbiology, 4th Ed.
Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!
Copyright 2000. Thomas M. Terry

  1. Today, look at phenomenon of GROWTH, how it is influenced by chemical & physical factors in environment.
  2. Understanding microbial growth is crucial to many industrial, health, and research considerations.
  3. For example: imagine you are hired by Pfizer to help develop new antibiotic from a strain of Bacillus. You have one 10,000 gallon tank available, costs $1000/day to run. You must decide how much inoculum to put in tank, when to harvest bacteria for maximum profit, minimum cost. How will you decide? What numbers do you need?
  4. Another example. You are hired as a research assistant in a laboratory. You need to grow bacteria to a concentration of 5 x 108 cells/ml, then harvest these cells for use in an experiment. When should you inoculate? How can you know when you have reached the desired cell concentration?

Batch vs. Continuous culture methods

Mechanics of batch growth.

Growth curve (see Fig. 6.1 in text)

4 phases: lag, exponential, stationary, and death

  1. Whylag phase?
    • Previous cells ran out of food, shut down many metabolic pathways needed for active growth, made adaptations necessary for dormancy and protection. Need to regenerate pools of essential nutrients before growth can resume, requires new enzyme synthesis and time for pathways to function.
    • Can reduce or eliminate lag phase by using cells that are not in stationary phase.
  2. Why exponential phase?
    • Cells in optimum growth state, divide repeatedly by binary fission at maximal rate.
    • Note: useful to calculate doubling time; can vary from 20 min to several days
  3. Why stationary phase?
    • Can be due to exhaustion of some critical nutrient, or to accumulation of waste products that slow down growth (e.g. acid buildup from fermentation).
  4. Why death phase?
    • Continued accumulation of wastes, exposure to oxygen, loss of cell's ability to detoxify toxins, etc. Note that death is exponential; 90% of cells die in certain time, another 90% in same time period, etc.
    • Note: in practice, try to prolong stationary phase, reduce death phase. Don't store cultures at room temperature on plates (ready exposure to oxygen, dessication, high temperature speeds oxidation reactions). Better, transfer to slants (tubes), store capped in refrigerator once grown. Still better, transfer to stab tube (soft nutrient agar), stopper and seal with airtight seal. Can recover viable culture even after a year of sitting on shelf!

Mathematics of growth; generation time

Growth equation

# of generations# of bacterialog2Nlog10N
0 1 or 20 0 0
1 2 or 21 1 .301
2 4 or 22 2 .602
3 8 or 23 3 .903
4 16 or 24 4 ...
5 32 or 25 5 ...
n 2n n .301n
growth equation Example:
measure culture at 9 a.m.: No = 10,000 cells/ml
measure culture at 3 p.m.: Nf = 100,000 cells/ml
calculate n = (5 - 4)/0.301 = 1/0.3 = 3.33 generations
total time = 6 hours = 360 minutes
360 minutes/3.33 generations = 108 minutes/generation
Conclude: generation time = 108 minutes
Note: be able to calculate g.t. Pay attention to units, ask if your answer is reasonable.

Group Practice

Graphical measurement of growth

See text fig. 6.2

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).

Measurement of growth

  1. Total Cell count
    • Petroff-Hausser chamber slide -- needs large conc. (107 cells/ml minimum) -- see Fig. 6.4
    • 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, etc.
  2. Viable count
    • This is typically carried out by 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.
    • CFU may or may not be same as number of cells --
    • Method is 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 won't 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.
  3. Optical 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. With less than 107 cells/ml, cannot detect bacteria.
      1. 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)
      2. Absorbance method
        • Use a spectrophotometer to accurately measure absorbance, usually at wavelengths around 400-600 nm.
        • Accurate measure of cells when concentration not too high. Easy and quick to measure (can sample in less than a minute)

Effects of temperature on growth

Different microbes adapted to different temperature ranges

Effects of oxygen on growth

Note: higher organisms all require oxygen; we are not used to thinking about other alternatives. But many microbes grow anaerobically some or all of the time.


  1. Obligate aerobes -- grow only when oxygen is present
  2. Facultative anaerobes -- grow with or without oxygen, grow better in oxygen (respire)
  3. Aerotolerant anaerobes -- ignore oxygen, grow equally well with or without
  4. Obligate anaerobes -- die in presence of oxygen
  5. Microaerophiles -- won't grow at normal atmospheric oxygen (20%), but require some oxygen for growth (2-10%)
Anaerobic habitats more common than expected. Ex: in human mouth, plaque contains bacterial zoo. Facultative anaerobes consume oxygen, create anaerobic microenvironment fit for obligate anaerobes.In general, wherever organic matter accumulates, microbes will use up oxygen faster than it can be replaced, create anaerobic environment. Esp. true under water, since oxygen is poorly soluble in water. Lakes and ponds stratify into aerobic (upper) and anaerobic (lower) zones in summer due to vigorous microbial growth on sediments.

Why obligate anaerobes?

Oxygen itself is reactive (oxidizing agent), capable of degrading organic molecules. But oxygen can easily generate very toxic byproducts, strong oxidizing agents that react indiscriminately with any organic molecules, including DNA, proteins, etc.:
Aerobes (and all cells able to tolerate oxygen) must have enzymes to get rid of these radicals. Superoxide dismutase and catalase are two crucial enzymes.
superoxide (O2-)+ H+ ---(superoxide dismutase)----> O2 + H2O2 peroxide (H2O2) ---(catalase)------> O2 + H2O
Note: If E. coli (a facultative anaerobe) is mutated so it loses these two enzymes, resulting mutant behaves like an obligate anaerobe -- good confirmation of idea.

Culture techniques

Effects of pH on growth

Diff microbes have diff pH optima:

Effects of solutes and water activity on growth

Effects of radiation on growth

Mechanisms of damage:

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