Lecture BIOD 7: Microbial Growth

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Lecture BIOD 7 Microbial Growth
- Growth is an increase in cellular constituents
that may result in an increase in cell size, an
increase in cell number, or both.
- When a microbiologist speaks of microbial
growth it is usually increase in cell number that
he/she is after.
- Consequently, there is a tendency for
microbiologists to follow microbial growth as
populations rather than following the growth of
individual cells.
- Microbiologists tend to be more interested in
population sizes than the size (mass) of any
individual cell.
- Typical measurement of microbial growth will be
done over the span of more than one microbial
generation.
- An increase in cell number is an immediate
consequence of cell division.
- Increase in cell numbers occurs when
microorganisms reproduce by a process like
budding or binary fission.
- Budding is a form of reproduction in which a
new cell is formed as an outgrowth from the
parent cell, as in the case of yeast and some
bacteria.
- The majority of bacteria reproduce by a
mechanism termed binary fission.
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- Binary fission generally involves the
separation of a single cell into two more or less
identical daughter cells, each containing, among
other things, at least one copy of the parental
DNA.
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• The Growth Curve

- The population growth is studied by analyzing
the growth curve of a microbial culture.
- The standard bacterial growth curve describes
various stages of growth a pure culture of
bacteria will go through, beginning with the
addition of cells to sterile media and ending
with the death of all of the cells present.
- Usually analyzed in a closed system called a
batch culture plotted as the logarithm of cell
number versus the incubation time.
- Bacteria added to fresh media typically go
through four more-or-less distinct phases of
growth lag, exponential, stationary, and death.

• Lag phase

- The period of apparent inactivity in which the
cells are adapting to a new environment and
preparing for reproductive growth.
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- Cells are usually synthesizing new components.
- In practice, bacteria from one medium to
another, where there are chemical differences
between the two media, typically results in a lag
in cell division.
- This lag in division is associated with a
physiological adaptation to the new environment.
- Cells may increase in size during this time,
but simply do not divide (by binary fission).
- Lag phase varies considerably in length
depending upon the condition of the
microorganisms and the nature of the medium.

• Log (exponential) phase

- The period in which the organisms are growing
at the maximal rate possible given their genetic
potential, the nature of the medium, and the
conditions under which they are growing.
- Cells in optimum growth state, divide
repeatedly by binary fission at maximal rate the
population doubles in every generation time.
- Generation time, also called Doubling time, is
the time it takes a bacterium to do one binary
fission starting from having just divided.
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- Generation times vary markedly with the species
of microorganism and environmental conditions
they can range from 10 minutes for a few bacteria
to several days with some eucaryotic
microorganisms.
- The population is most uniform in terms of
chemical and physical properties during this
period.

• Stationary phase

- Eventually population growth ceases, and the
growth curve becomes horizontal.
- Increase in cell number due to cell divisions
exactly balanced by a decrease in cell number due
to death.
- Cell death may result from Nutrient limitation
Toxic waste accumulation (e.g. acid buildup
from fermentation) as well as O2 depletion,
critical population level reached.

• Death phase

- Stationary phase, in a standard bacterial
growth curve, is followed by a die-off of cells,
called Death phase.
- It is the period in which the cells are dying
at an exponential rate.
- Some of the reasons are continued accumulation
of wastes, loss of cell's ability to detoxify
toxins, etc.
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• The Algebra of Exponential Growth

- Microbial growth can be described by certain
mathematical terms Mean Generation Time and Mean
Growth Rate.
- Mean generation (doubling) time is the time
required for the population to double.
- Mean growth rate constant is the number of
generations per unit time, often expressed as
generations per hour.

• Growth equation

• n number of generations.
• Nf final conc. of cells (e.g. 109/ml).
• No initial conc. of cells (e.g. 103/ml)

Nf 2n N0
Example
- Calculate Generation time and Growth rate?
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• Measurement of Microbial Growth

- There are many ways to measure microbial
growth (growth rates generation times).
- Population mass or number may be followed
growth increases both.
- Direct cell count methods and may be
accomplished by direct microscopic observation on
specially etched slides (such as Petroff-Hausser
chambers or hemocytometers) or by using
electronic counters.
- Electronic counters, such as Coulter Counters,
count microorganisms as they flow through a small
hole or orifice.
- Direct cell count methods do not distinguish
between living and dead cells.
- Viable cell counts involve plating diluted
samples (using a pour plate or spread plate) onto
suitable growth media and monitoring colony
formation this type of method counts only those
cells that are reproductively active.
- It is typically carried out by Colony Forming
Units (CFU) 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.

- Spread plate bacteria are spread on the
surface of agar using some sterile spreading
device.
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- Pour plate bacteria are mixed with melted agar
and cooled colonies grow throughout the agar.
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- Absorbance method it is one of the optical
methods for counting cells can estimate cell
numbers accurately by measuring visible
turbidity. Light scattered is proportional to
number of cells.
- 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
measure a sample in less than a minute).
- Measurement of cell mass may be used to
approximate the number of microorganisms if a
suitable parameter proportional to the number of
microorganisms present is used.
- Suitable parameters may be dry weight, light
scattering in liquid solutions, or biochemical
determinations of specific cellular constituents
such as protein, DNA, or ATP.

• Balanced and Unbalanced Growth

- Balanced (exponential) growth occurs when all
cellular components are synthesized at constant
rates relative to one another.
- Unbalanced growth occurs when the rates of
synthesis of some components change relative to
the rates of synthesis of other components. This
usually occurs when the environmental conditions
or nutrient levels change.
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• The Influence of Environmental Factors on Growth

- Most microorganisms only grow in fairly
moderate environmental conditions, but some,
referred to as extremophiles, can grow under
harsh conditions that would kill most other
organisms.
- The kinds of microorganisms found in a given
environment and the rates at which they grow can
be influenced by a variety of factors, both
physical and biochemical.
- Physical factors include pH, temperature,
oxygen concentration, moisture, hydrostatic
pressure, osmotic pressure, and radiation.
- Nutritional (biochemical) factors include
availability of carbon, nitrogen, sulfur,
phosphorus, trace elements, and, in some cases,
vitamins.

• Solutes and Water Activity

- Cells require certain amount of free water to
be able to carry out metabolism.
- Water activity (aw ) is used as a quantitative
measurement of the availability of water.
- Microorganisms are separated from their
environment by a selectively permeable PM? they
are affected by changes in osmotic concentration
of their surroundings.
- The availability of water is inversely related
to osmotic pressure.
- Osmosis is the movement of water into a cell by
simple diffusion occurs from regions of high
water concentration to regions of low water
concentration.
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- A hypotonic solution is one that has a lower
solute concentration than the cell's cytoplasm.
- if the cell is placed in a hypotonic
solution?water enters cell?cell bursts.
- A hypertonic solution is one that has a higher
solute concentration than the cell's cytoplasm.
- if the cell is placed in a hypertonic
solution?water out of cell? cytoplasm shrink and
pulls out of cell (phenomenon called plasmolysis).
- Osmotolerant organisms can grow in solutions of
both high and low water activity.
- Halophiles require environments of low water
activity (high osmotic pressure) in order to
grow. Ex. Halobacterium halobium grows in Dead
Sea, Great Salt Lake, evaporating salt flats.
- Microorganisms growing in a habitat with low
water activity (aw) usually maintain a high
internal solute concentration in order to retain
water.

• pH

- pH is the negative logarithm of the hydrogen
ion concentration.
- The range over which most organisms can grow
tends to vary over no more than a single pH unit
in either direction (e.g., from pH 6 to pH 8 for
an organism whose pH optimum is pH 7).
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- Microorganisms are classified as

• Acidophiles grow best between pH 0 and 5.5
• Neutrophiles grow best between pH 5.5 and 8.0
• Alkalophiles grow best between pH 8.5 and 11.5
• Extreme alkalophiles grow best at pH 10.0 or
  higher

- Despite wide variations in habitat pH, the
internal pH of most microorganisms is maintained
near neutrality either by proton/ion exchange or
by internal buffering.

• Temperature

- Temperature has a profound effect on
microorganism viability, primarily because
enzyme-catalyzed reactions are sensitive to
temperature.
- At low temperatures, a temperature rise
increases the growth rate by increasing the rate
of enzyme reactions.
- At high temperatures, microorganisms are
damaged by enzyme denaturation, membrane
disruption, and other phenomena.
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- Microorganisms exhibit distinct cardinal
temperatures (minimal, maximal, and optimal
growth temperatures).
- For most bacteria, the range from minimum to
maximum is about 30C (e.g., from 44C down to
14C).
- Psychrophiles can grow well at 0C, have
optimal growth at 15C or lower, and usually will
not grow above 20C.
- Mesophiles have growth minima of 15 to 20C,
optima of 20 to 45C, and maximum of about 45C
or lower.
- Thermophiles have growth minima around 45C,
and optima of 55 to 65C.
- Hyperthermophiles have growth minima around
55C and optima of 80 to 110C.
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• Oxygen

- Microorganisms differ in their requirements of
molecular oxygen (i.e., O2) as well as other
atmospheric gasses (e.g., carbon dioxide).
- Obligate aerobes are completely dependent on
atmospheric O2 for growth.
- Facultative anaerobes do not require O2 for
growth, but do grow better in its presence
- Aerotolerant anaerobes ignore O2 and grow
equally well whether it is present or not.
- Obligate (strict) anaerobes do not tolerate O2
and die in its presence.
- Microaerophiles are damaged by the normal
atmospheric level of O2 (20) but require lower
levels (2 to 10) for growth.
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• Pressure

- Most microorganisms live on land or surface of
water no effect of pressure.
- Some bacteria can survive in deep oceans with
pressures up to 600 1100 atm.
- Barotolerant organisms are adversely affected
by increased pressure, but not as severely as are
non-tolerant organisms.
- Barophilic organisms require, or grow more
rapidly in the presence of, increased pressure.

• Radiation

- Ultraviolet radiation damages cells by causing
the formation of thymine dimers in DNA.
- Ionizing radiation such as X rays or gamma rays
are even more harmful to microorganisms than
ultraviolet radiation.

• Growth in Natural Environments

- Microorganisms will grow in microenvironments
until an environmental or nutritional factor
limit this process.
- These factors include water, energy,
temperature, nutrients, pressure, pH, salinity
etc.
- Limiting factors can change over time and space.
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• Liebigs Law of the Minimum

- The total biomass of an organism will
determined by the nutrient present in the lowest
concentration relative to the organism
requirements.

• Shelfords Law of Tolerance

- There are limits to environmental factors below
and above which a microbe cannot survive and
grow, regardless of nutrient supply.