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Tortoraetal.Chap6
Microbial Growth
Definitions of Growth
Steady increase in all the chemical components of an organism that may result in an increase
cell size, cell number or both
A. Chemical factors
Nutrients are substances used in biosynthesis and energy release and are therefore required
for growth
One must define nutritional requirements in order to cultivate the microbe in the laboratory
Chemical factors are supplied by i) the culture medium (pl. - media) that contains substrates
required for growth and ii) culture conditions (i.e., aerobic vs anaerobic conditions).
Carbon
o Life on earth is carbon based
o Half of the dry weight of a typical cell is carbon
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Nitrogen
o Nitrogen makes up approximately 14% of the dry weight of a typical cell
o Major constituent of protein and nucleic acids, some carbohydrates and lipids
o NH3, NO3-, N2 (nitrogen fixation) and organic N compounds (e.g., amino acids) from the
environment. Some bacteria use atmospheric nitrogen (N2) as a nitrogen source
Phosphorus
o component of phospholipids and nucleic acids, nucleotides such as ATP, some proteins
o available as organic and inorganic forms in the environment
Sulfur
o structural role in methionine and cysteine as well as a number of vitamins (thiamine,
biotin), coenzyme A and some carbohydrates
o available usually from inorganic sources SO42- or H2S and organic sulfur compounds such
as cysteine
K, Ca, Mg, and Fe are cations in cells and required for a variety of roles
e.g., - cofactors (K+, Ca2+, Mg2+, and Fe2+ or Fe3+)
- stabilize membranes and ribosomes (Mg2+)
- contribute to heat resistance of endospores (Ca2+)
- components of biomolecules such as cytochromes (Fe2+ and Fe3+)
3. Oxygen
a) Aerobic organisms
growth at full atmospheric O2 tensions (21% O2 in the atmosphere)
facultative organisms (under appropriate nutrient and culture conditions) can grow under
either aerobic or anaerobic condition
obligate aerobes - require O2 for growth
O2 is poorly soluble - forced aeration is often used in culture systems to provide O2
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b) Anaerobic organisms
obligate (strict) anaerobes - grow only in the absence of O2; sensitive to O2 and brief exposure
will kill these organisms; perhaps because these organisms are unable to detoxify some of the
products of O2 metabolism
lack a respiratory system and cant use oxygen as a terminal electron acceptor
These organisms do use oxygen found in cellular materials
Catalase
o destroys H2O2
o H2O2 + H2O2 2 H2O + O2
o Catalase test - 30% H2O2 place on cells. Cells with catalase activity produces vigourous
bubbling as O2 is released
Peroxidase
o destroys H2O2 but does not produce O2. May require a reductant such as NADH
o H2O2 + 2H+ 2 H2O
Superoxide reductase
o Found in some obligately anaerobic prokaryotes
o O2.- + 2H+ + cytochrome creduced H2O2 + cytochrome coxidized
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c) Aerotolerant anaerobes
tolerate O2 and grow in its presence even though they cant use oxygen.
Aerotolerant organisms can tolerate oxygen because they produce SOD or equivalent system that
neutralizes toxic oxygen species. Usually lack catalase activity
d) Microaerophiles
grow only at reduced O2 concentrations (2 to 10%)
These organisms have limited capacity to respire or have some oxygen-labile molecules;
sensitivity to oxygen may also be due to the sensitivity superoxide radicals and peroxides
O2 usually excluded from culture systems by one or a combination of the following mechanisms
Fill container to the top and seal
Boil medium to drive out O2
Use reducing agents that react with O2; reduces it to H2O (e.g., thioglycolate, cysteine, H2S)
Seal containers under O2 free gas
Use redox indicators such as resazurin to indicate the presence of O2.
Use O2 consuming devices (catalyst)
Work under a stream of O2 free gas or in an anoxic glove box/anaerobic chamber
5. Growth Factors
Some microbes have the enzymes and biochemical pathways needed to synthesize all cellular
components using minerals and sources of energy, carbon, nitrogen, phosphorus and sulfur.
Other microbes lack one or more enzymes necessary to synthesize essential constituents they get
these constituents or precursors from the environment
Growth factors are organic compounds that are essential cellular components or precursors of these
components but cannot be synthesized by the organism
Major Classes of Growth factors
1. amino acids
2. purine and pyrimidines
3. vitamins (e.g., thiamine, biotin, cobalamin, pyridoxine)
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o Bioassays using microbes to detect the specific growth factor that they need. Growth-
response assay uses this approach to detect the amount of a growth factor in solution.
These assays can be specific, sensitive, simple and quantitative
o Manufacture of growth factors by specific microorganisms (e.g., Vitamin D by
Saccharomyces) in industrial fermentations
B. Physical (or environmental) Factors
b) Optimum temperature
highest rate of growth and reproduction, always nearer maximum temperature
i) Psychrophiles
Grow well at 0C and have an optimum temperature 15C and a maximum temperature
around 20C
heat sensitive and unable to survive temperate climates
Adaptations to Psychrophily
o Enzymes, transport systems and protein synthetic apparatus work well at low
temperatures
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o greater amounts of polar amino acids and lesser amounts of hydrophobic amino acids
iii) Mesophiles
Optimum temperature between 25 and 40C
Minimum temperature between 15 and 20C
Maximum temperature 45C
Most common type of microbe
e.g., E. coli
Optimum temperature < 39C
Maximum temperature < 48C
Minimum temperature 8C
iv) Thermophiles
Optimum temperature between 50 and 60C
Minimum temperature around 45C
Maximum temperature 45C
v) Hyperthermophiles
Optimum temperature > 80C
Extreme thermophiles are usually Archaea
The highest growth temperatures for an archaeon is 113C (Pyrolobus fumarii)
Adaptations to Thermophily
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The presence of certain solutes such as di-inositol phosphate and diglycerol phosphate
Applications of Thermophily
High temperature enzymes
e.g., feed pelleting process
PCR Taq DNA polymerase from Thermus aquaticus
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Intracellular pH
o Intracellular pH is usually between pH 6 to 8 but internal pH as low as 4.6 and as high as
9.5 have been measured
o Maintained by pumping H+ across the membrane, internal buffering and synthesizing new
proteins (e.g., acid shock proteins and heat shock proteins) that function by pumping
protons or acting as chaperones
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a) Halophilic bacteria
A organism requiring salt (NaCl) for growth
microbes found in the sea (which is 3% NaCl) usually have a growth requirement for salt
Halotolerant organisms can withstand some reduction in aw but generally grow best without added
solute
Osmotolerant grow over a wide range of water activity
Osmophiles - require high solute (e.g., sugar) concentration for growth
Xerophiles able to grow in very dry environments (i.e., made dry by lack of water)
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Liebigs law - the total biomass of an organism will be determined by the nutrient present in the
lowest concentration relative to the organisms requirements
Shelfords law there are limits to environmental factors below and above which a
microorganism cannot survive and grow regardless of the nutrient supply
Most bacteria are likely to experience starvation. How do they deal with nutrient limitation?
Biofilms
Most microbes are typically found in biofilms in nature
Biofilms consist of cells embedded in EPS (Chapter 4)
Microbes in biofilms share nutrients, communicate (e.g., quorum sensing), exchange genetic
information and are sheltered from adverse environmental factors (i.e., desiccation,
antibiotics, host immune response)
Microbes in biofilms can be 1000X more resistant to antimicobial compounds
Microbes in biofilms can carry out complex chemical processes (i.e., breakdown of plant cell
walls such as occurs in the rumen)
Inoculum (pl. = inocula) = microbes introduced into a culture medium to initiate growth. These
cells multiply and are referred to as the culture.
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Fastidious microorganisms - have very rigorous or complex requirements (e.g., for vitamins,
amino acids...)
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2. Complex media
certain components are of unknown composition and these components may change from
batch to batch.
Use of this type of medium results in the loss of control of nutrient composition
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Selective medium
A medium with a composition favoring growth of certain types of microorganisms while
inhibiting growth of any other microorganisms that may be present.
Examples
Differential medium
A medium that contains substance(s) that permits for the differentiation of particular
metabolic activities during growth. Useful in distinguishing particular groups of
microbes and may provide information useful in identification
Examples
Examples
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C. Enrichment technique
Developed by Beijerinck
The use of culture media or conditions that favour growth of one type or group of
physiologically related microorganisms over all other microorganisms present in the sample
Aseptic Technique
Series of steps used to minimize contamination during the manipulations of cultures and
sterile culture media
Sterilize all media and implements for handling materials of interest
Clean working area
Limit exposure to potential sources of contamination
2. Glycerol stocks
Sterile glycerol is added to liquid cultures to a final concentration of 15 25%
The stocks are placed in small plastic tubes with tight fitting lids (i.e., preferably screw cap
tubes with gaskets in the lids)
The glycerol stocks are stored at -20C (1 to 2 years) or -80C (up to 10 years or more)
3. Lyophilization
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Freeze drying
Culture is quick-frozen at temperatures ranging from -50 to -90C and then dried under
vacuum on a lyophilizer; freeze dried cultures are stored in sealed glass ampules for extended
periods of time.
Microbial Culture Collections
Sources of microbial cultures
Cultures are distributed for a fee or free depending on the culture collection
A. Population Growth
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Growth rate
change in cell number or cell mass per unit time
Generation
interval for the formation of two cells from one cell
Growth by binary fission results in exponential growth of the population (Figure 6.13 & 6.14)
21222324
(1) Nt = N02n
Growth rate can also be expressed as the mean growth rate constant (k). The specific growth rate
is a measure of the number of generations that occur per unit time
Can now calculate the mean generation time (g) or mean doubling time.
When the population doubles t = g and Nt = 2N0; substitute 2N0 into (3)
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Therefore
(5) g = 1/k
Generation time can also be calculated from the slope of a line obtained in a semi-log plot of
exponential growth
2. Culture Systems
"Fermentation" - cultivation of microorganisms in a controlled, enclosed system
i. Batch Culture
A fixed volume of liquid medium is inoculated and incubated for an appropriate period of time
with no further addition of microorganisms or growth substrates
closed environment
most common method of microbial cultivation
nutrient concentration is a determinant of growth rate and cell yield
Ultimately the culture quits growing due to nutrient limitation or product accumulation
Open system
system can be manipulated to reach an equilibrium or steady state where the cell density and
nutrient status remain constant
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Can control culture growth rate as well as yield of cells by manipulating dilution rate and the
level of the limiting nutrient, respectively
More sophisticated apparatus required
Superior productivity possible because of reduced downtime.
e.g., Chemostat
uses dilution rate and nutrient concentration to control growth and population density
growth rate (adjust dilution rate) and yield (adjust limiting nutrient) can be controlled
independently of each other
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i) Lag Phase
Initial phase during which time cells are adjusting their metabolism to prepare for a new cycle of
growth.
There is no increase in cell number - increase in cell size
The cells are transporting nutrients, synthesizing RNA and subsequently enzymes needed for
growth; replicating DNA
The length of this phase depends on the history of the culture and growth conditions
Examples:
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Total number of viable cells remains unchanged because i) growth rate = death rate (i.e.,
some cells in the population grow while others die. This is known as cryptic growth) or ii)
the population may not be dividing but remain metabolically active
Stationary phase is entered because 1) nutrient limitation, 2) oxygen limitation, 3) build up of
toxic wastes (e.g., organic acids), 4) a critical population level is reached, or 5) several of
these factors acting together
i) Not all cells are culturable = Viable but nonculturable (VBNC) cells.
Cells are viable as demonstrated by the presence of metabolic activities but can't be
cultivated in the lab - detected by discrepancies between indirect and direct counts. VBNC
cells are genetically programmed to become dormant (genetic response triggered in starving
stationary phase cells) and when appropriate conditions become available (e.g., change in
temperature, passage through animals), the cells begin growing again.
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ii) Programmed cell death. A fraction of the microbial population is genetically programmed
to commit suicide nonculturable cells are dead and the nutrients that they leak enable
eventual growth of those cells in the population that did not commit suicide.
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4. Measurement of Growth
Enumeration of microbial populations or measuring mass
Advantages
rapid
counts all cells in a sample (can often count individual cells in clumps)
can acquire cell morphology information with these methods
Disadvantages
can't determine which cells are viable unless they are treated in a special manner
(e.g.,fluorescent live/dead cell stains).
small cells are difficult to see
affected by debris in samples
not suitable for cell suspensions of low density (< 106/mL); precision difficult to achieve
motile cells are difficult to count
phase contrast microscopy required if sample not stained
may require expensive pieces of equipment
unable to perform further studies on the observed microbes without further cultivation
Filtration
known volume of a suspension filtered onto a black polycarbonate filter membrane.
cells are stained with fluorescent dyes and counted under the microscope
Coulter Counter
automated method of counting cell.
as cell pass through a aperture they disturb an electric field
perturbations are transformed into number and size data.
Most useful for larger cells
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takes time for data acquisition (i.e., Cells must grow for >12 h to be counted with the
viable count methods
size of colonies vary and it is easy to miss small colonies
subject to large errors if not done carefully require adequate replication
Most Probable Number (MPN)
another technique for counting viable CFU
dilute to extinction - such that not all aliquots transferred to tubes of growth medium will
contain a cell
following incubation one checks for growth and compares results to a table of statistical
probability for obtaining the observed results.
Membrane filtration
Aquatic samples are filtered through a membrane trapping cells on the membrane
The membrane is placed on an agar medium and incubated until each cell forms a colony
Useful for analyzing water samples especially when the populations are low
Turbidity (Spectophotometry)
rapid and sensitive method for obtaining estimate of culture density
The more cells that are present the more light that is scattered by a suspension
can measure transmittance of light and determine the optical density (OD) of a suspension
using a spectrophotometer
growth results in increased turbidity and OD proportional to cell number for unicellular
organisms
Can generate a standard curve to relate OD to CFU's/unit volume or some other measure of
growth (e.g., dry weight)
Metabolic Activity
Measures a metabolic product and assumes there is a direct relationship between the amount
of the metabolic product and the cell number.
Measurement of CO2 evolution
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