Transcript Document

Microbial Growth
Environmental influences and adaptations
Bio3124
Lecture #5
Growth
• Under favorable nutritional conditions
• Biosynthesis leads to increase in cellular
constituents
• Cells divide and population increases
• Growth: increase in population size not
individual cells size
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Binary Fission and Exponential Growth
20=1
1st division
2nd division
21=2
22=4
3rd division
23=8
4th division
24=16
N = No . 2n
n = number of divisions (generations)
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The Mathematics of Growth
Two parameters are important
1. Generation time (g)
• time required for the population to double in size
– Varies depending on species of microorganism and
environmental conditions
– range is from 10 minutes for some bacteria to several
days for some eukaryotic microorganisms
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The Mathematics of Growth
2. Mean growth rate constant (k)
• Mean number of generations (divisions) per hour
– This shows how fast the cells are growing under culture
conditions during log phase
– it is the inverse of “g” value
– ie. k=1/g (hr-1)
• How do you find the generation time?
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The Mathematics of Growth
• N0 cells after “n” generations
produce N cells over a given
incubation time (t)
• Since population increases
exponentially,
• then the final cell yield is an
exponential function,
N= N02n
log N= log (N0 . 2n )
log N= log N0+ n.log2
n.log2=log N-log N0
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The Mathematics of Growth
• Solving for n
(number of generations)
n = (log N - log N0)/log2
n = (log N - log N0)/0.301
• Since “n” generations happened
over “t” incubation time
Then the mean generation time:
g=t/n (min or hr)
• Graphically this corresponds to the time interval that
cells doubled in the linear portion of log phase.
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Problem
A bacterial culture grows from 1 X 106 cells/ml to 6.4 X 107
cells/ml in 2 hours. What is the generation time, number of
generations and the average growth rate constant of this
bacterial culture?
N0= 1 X 106 , N= 6.4 X 107, t=120 min
n = (log N - log N0)/0.301
n = (log 6.4 X 107 - log 1 X 106 )/0.301
n= 6 generations
G=120/6
G=20 min or 0.33 hr
K=1/g
K=1/0.33
K=3 generations per hour
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Experimental Growth Curve
• Plot of population vs time
• batch culture
– culture incubated in a closed vessel with a single
batch of medium
• usually plotted as logarithm of cell number
versus time
• Has four distinct phases
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Growth Curve
Has four distinct phases
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Lag Phase
• cells synthesizing new components
– reorganizing gene expression
– adapt to new medium
• varies in length
– in some cases can be very short or even
absent
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Exponential Phase
• also called log phase
• rate of growth is constant
• population is most uniform in terms of
chemical and physical properties during
this phase
• Balanced growth: cellular constituents
manufactured at constant rates relative to
each other
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Limiting nutrient concentration
Yield: function of the availability of limiting nutrient
Cellular
Mass
Time
Total Growth
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Stationary Phase
• Total number of viable cells remains
constant
– may occur because metabolically active cells
stop reproducing
– may occur because reproductive rate is
balanced by death rate
• cells are smaller
• remodeling of gene expression
• secondary metabolites produced
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Possible reasons for entry into
stationary phase
•
•
•
•
nutrient limitation
limited oxygen availability
toxic waste accumulation
critical population density reached
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Starvation responses
• decrease in size, protoplast shrinkage, and
nucleoid condensation
• production of starvation proteins
– Chaperones prevent protein denaturation
– DBPs (DNA binding proteins) protect DNA
– Increased PG cross-linking strengthens cell
wall
• Activation of mechanisms for long-term survival
– increased virulence
– morphological changes e.g., endospore
formation and differentiation
• Spore bearers: Genera Bacillus and Clostridium
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Sporogenesis
• Also called
endospore
formation or
sporulation
• normally
commences
when growth
ceases because
of lack of
nutrients
• Is a complex
multistage
process
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Sporulation
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Death Phase (decline)
• Two alternative hypotheses
– Cells are Viable But Not Culturable (VBNC)
• Cells alive, but dormant
• programmed cell death
– Fraction of the population genetically programmed to
die (commit suicide)
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Prolonged Decline in Growth
• bacterial population
continually evolves
• process marked by
successive waves of
genetically distinct
variants
• natural selection occurs
• Secondary metabolites
– Antibiotics
– Modified amino acids
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Measurement of
Microbial Growth
• can measure changes in number of cells in a
population
• can measure changes in mass of population
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Measurement of Cell Number
• Direct cell counts
– counting chambers
– electronic counters
– collecting on filter membranes and staining
with fluorescent dyes
• Viable cell counts
– plating methods
– membrane filtration methods
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Counting chambers
•
•
•
•
Petroff-Hausser chamber or Hemocytomer
easy, inexpensive and quick
useful for counting both eukaryotes and prokaryotes
cannot distinguish living from dead cells
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Electronic counters: Coulter counter
• useful for large microorganisms
• Less sensitive for bacteria
• microbial suspension forced through small
orifice
• movement of microbe through orifice impacts
electric current that flows through orifice
• instances of disruption of current are counted
• Can’t tell the dead and live apart
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Direct counts on membrane filters
• cells are stained with
fluorescent dyes
• cells filtered through special
membrane that provides dark
background for observing cells
• useful for counting bacteria
• with certain dyes, can
distinguish live from dead cells
• Propidium iodide (dead cells,
red)
• Syto-9 (live cells, green)
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Enumeration: Viable Counting Methods
• spread and pour plate techniques
– diluted sample of bacteria is spread over solid
agar surface or mixed with agar and poured into
Petri plate
– after incubation the numbers of organisms are
determined by counting the number of colonies
multiplied by the dilution factor
– results expressed as colony forming units per
volume (CFU/ml)
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Plating methods…
• Pour-plate or Spread-plate
• simple and sensitive
• widely used for viable counts of microorganisms
in food, water, and soil
• inaccurate results obtained if cells clump
together
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Another Viable Count Method – growing on Membrane filters
Especially useful for analyzing aquatic samples
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Measurement of Cell Mass
• Dry weight
– time consuming and not very sensitive
• Quantity of a particular cell constituent
– e.g., protein, DNA, ATP, or chlorophyll
– useful if amount of substance in each cell is constant
• Turbidometric measures (light scattering)
– quick, easy, and sensitive
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Spectrophotometry:
more cells

more light
scattered

less light
detected
Cannot distinguish between dead and live cells
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The Influence of Environmental
Factors on Growth
• most organisms grow in fairly moderate
environmental conditions
• extremophiles
– grow under harsh conditions that would kill
most other organisms
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Temperature
• organisms
exhibit distinct
cardinal growth
temperatures
– minimal
– maximal
– optimal
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Temperature ranges for microbial growth
(Pathogens)
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Adaptations of thermophiles
• protein structure stabilized by a variety of
means
– e.g., more H-bonds, hydrophobic core
– e.g., more proline= less flexibility
– e.g., chaperones
• histone-like proteins stabilize DNA
• membrane stabilized by variety of means
– e.g., more saturated, more branched and higher
molecular weight lipids
– e.g., ether linkages (archaeal membranes)
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Classification of bacteria on the basis of their Oxygen need
need
oxygen
prefer
oxygen
ignore
oxygen
oxygen is
toxic
< 2 – 10%
oxygen
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Oxygen is toxic
• oxygen easily reduced to toxic products
that oxidize cellular components
– superoxide radical (O2+e-→O2-)
– hydrogen peroxide (O2-+e-+2H+ →H2O2)
– hydroxyl radical (H2O2+ e-+H+ →H2O+OH·)
• aerobes produce protective enzymes
– superoxide dismutase (SOD)
2O2-+2 H+ → O2+H2O
– Catalase
2H2O2 → O2+ 2H2O
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Solutes and Water Activity
• water activity (aw)
– amount of water available to cell
– Inversely related to osmotic pressure
– higher [solute]  lower aw
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Adaptations: effect of NaCl on microbial growth
• Nonhalophiles grow 0.1-1 M
• Halophiles
• grow optimally at >0.2 M
• Moderate halophiles
– Optimal growth at ~2M
• extreme halophiles
– require >2 M
• Adaptation: Know how to
control the water activity
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Control of water activity
• In hypertonic environments many use compatible solutes to
increase their internal osmotic concentration ie. reduce aw
(eg. amino acids, choline, K+)
• In hypotonic environment, release solute from internal
environment by opening channels through signaling by a
mechanoreceptor sensor protein
• Water can cross the cell membrane either by diffusion or
more quickly using aquaporins
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pH
• Measure of H+ concentration
– pH range : 0.0 – 14
• Optimal pH for growth:
–Acidophiles : pH 0.0 – 5.5
–Neutralophiles : pH 5.5 – 8.0
–Alkaliphiles : pH 8.5 – 11.5
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Effects of pH
• Extreme pH
– Loss of enzymatic activity; denaturation and
degradation of proteins.
– Hydrolysis of DNA and RNA
– Loss of membrane integrity
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pH Homeostasis
• Strategy: maintain an
internal pH near
pH,2
neutrality
pH,9
• Synthesize proteins,
pH,5
provide protection
– e.g., acid-shock
proteins to maintain
the normal protein
folding
Transporters balance internal H+
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