Transcript microbial growth

MICROBIAL GROWTH
The reproductive strategies of eukaryotic
microbes
◦ asexual (mitosis) and sexual (meiosis)
 Bacteria and Archaea
◦ asexual - binary fission, budding,
filamentous
◦ all must replicate and segregate the
genome prior to division
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increase in cellular constituents that may
result in:
◦ increase in cell number
◦ increase in cell size
 growth refers to population growth rather
than growth of individual cells
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Population growth – studied by analyzing
the growth curve of a microbial culture
 observed when microorganisms are
cultivated in batch culture
◦ culture incubated in a closed vessel with
a single batch of medium
◦ No in n out fresh medium, nutrient conc
decline, waste increase
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usually plotted as logarithm of cell number
versus incubation time
 has four distinct phases
◦ lag, exponential, stationary, senescence,
and death
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Microorganism introduced into fresh medium
No increase in cell number
cell synthesizing new components
◦ to replenish spent materials
◦ to adapt to new medium or other conditions
◦ time to recover; cell may be injured
varies in length
◦ in some cases can be very short or even
absent
also called log phase
 Microorganisms are growing & dividing at
max rate
 rate of growth and division is constant and
maximal (completing cell cycle,doubling)
 population is most uniform in terms of
chemical and physical properties during
this phase
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during log phase, cells exhibit balanced
growth
◦ cellular constituents manufactured at
constant rates relative to each other
Unbalanced growth
 rates of synthesis of cell components vary
relative to each other until balanced state is
reached
 occurs under a variety of conditions
◦ change in nutrient levels
 shift-up (poor medium to rich medium)
 shift-down (rich medium to poor
medium)
◦ change in environmental conditions
At sufficiently high nutrient concentration, transport system are saturated,
growth rate does not rise further with increasing nutrient conc.
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closed system population growth eventually
ceases, growth curve become horizontal
Happened at population level around 109 cell
per ml
Final population size depend on nutrient
availability, type of microorganisms
total viable cell number remain constant
◦ active cells stop reproducing or
reproductive rate is balanced by death rate
nutrient limitation
(nutrient deplete, population growth will slow)
 limited oxygen availability
(O2 deplete, only surface have adequate O2)
 toxic waste accumulation
(byproduct toxic to microbe)
 critical population density reached
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entry into stationary phase due to starvation
and other stressful conditions activates
survival strategy
◦ morphological changes
 e.g., endospore formation
◦ decrease in size, protoplast shrinkage,
and nucleoid condensation
◦ RpoS protein assists RNA polymerase in
transcribing genes for starvation proteins
production of starvation proteins
◦ increase cross-linking in cell wall
◦ Dps protein protects DNA
◦ chaperone proteins prevent protein
damage
 cells are called persister cells
◦ long-term survival
◦ increased virulence
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No of viable cell decline at exponential
rate
 Nutrient deprivation, buildup of toxic waste
cause irreparable harm to cell
 No cellular growth if transfer to fresh
medium
 Because loss viability not accompany by
loss in total cell no. assume that cell died
but not lyse
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two alternative hypotheses:
a) cells are Viable But Not Culturable (VBNC)
 Cells are unable to grow temporarily
 cells alive, but dormant, capable of new
growth when conditions are right
b) programmed cell death
• fraction of the population genetically
programmed to die (commit suicide)
• Some cell die, nutrient they leak enable
growth of other cell
• Altruistic – they sacrifice themselves for
benefit of others
Exponential phase – microbe dividing at
constant interval
 generation (doubling) time
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◦ 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
Many ways to measure growth rate and
generation time
 can measure changes in number of cells
in a population
 can measure changes in mass of
population
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direct cell counts
◦ counting chambers
◦ on membrane filters
haemocytometer
 easy, inexpensive, and
quick
 useful for counting
both eukaryotes and
prokaryotes
 cannot distinguish
living from dead cells
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On bottom of chamber is an etched grid to
facilitate counting the cells
 Number of microbe calculated by taking
into account the chamber’s volume and
dilution made
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Sample is filtered through black
polycarbonate membrane that provides
dark background for observing cells
 cells are stained with fluorescent dyes
 Observed microscopically and count
 useful for counting bacteria (aquatic
sample)
 with certain dyes, can distinguish living
from dead cells
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Count only those able to reproduce when
cultured (plate count)
 Simple, sensitive
 Viable counting method
- spread plate, pour plate
- membrane filtration
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spread and pour plate techniques
◦ diluted sample of bacteria is spread over
solid agar surface or mixed with agar and
poured into Petri plate
◦ Cell with grow as distinct colony
◦ 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 (CFU)
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membrane filter technique
◦ bacteria from aquatic samples are
trapped on membranes of known pore
size
◦ membrane soaked in culture media
◦ colonies grow on membrane
◦ colony count determines number of
bacteria in original sample
Measure cell mass can be used to follow
growth
 dry weight
 turbidometric measures
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Dry weight
 Cell growing in liquid media – centrifuge,
washed – dried in oven – weight
 Measure growth of filamentous fungi/
bacteria
 Time consuming, not very sensitive
turbidometric measures
 Micobial cell scatter light that strike them
 Amount of scattering proportional to the
biomass of cell present
 Extent of light scattering can be measure
using spectrophotometer (absorbance)
 Increase cell concentration, greater
turbidity, more light scattered and
absorbance reading will increase
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Batch culture(closed system)
- nutrient not renewed
- waste not removed
- exponential growth last only for few generation
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growth in an open system (continuous
culture system)
- continual provision of nutrients
- continual removal of wastes
- maintains cells in log phase at a constant
biomass concentration for extended periods
constant supply of cells in exponential
phase growing at a known rate
 study of microbial growth at very low
nutrient concentrations, close to those
present in natural environment
 study of interactions of microbes under
conditions resembling those in aquatic
environments
 food and industrial microbiology
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rate of incoming medium =
rate of removal of medium
containing microorganisms
from vessel
an essential nutrient is in
limiting quantities
Growth rate determined by
rate at which fresh medium
fed into chamber
Has photocell to measure turbidity
 flow rate of media automatically regulated
to maintain a predetermined turbidity or
cell density
 no limiting nutrient (nutrients in excess)
 Turbidostat maintain desired cell density
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most organisms grow in fairly moderate
environmental conditions
 extremophiles
◦ grow under harsh conditions that would
kill most other organisms
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Solute & water activity, pH, temperature,
oxygen level
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changes in osmotic concentrations in the
environment may affect microbial cells
◦ hypotonic solution (lower osmotic
concentration)
 water enters the cell
 cell swells may burst
◦ hypertonic (higher osmotic concentration)
 water leaves the cell
 membrane shrinks from the cell wall
(plasmolysis) may occur
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halophiles
◦ grow optimally in the presence of NaCl or
other salts at a concentration above about
0.2M
extreme halophiles
◦ require salt concentrations of 2M and 6.2M
◦ extremely high concentrations of potassium
◦ cell wall, proteins, and plasma membrane
require high salt to maintain stability and
activity
measure of the
relative acidity of a
solution
 negative logarithm
of the hydrogen
ion concentration
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acidophiles
◦ growth optimum between pH 0 and pH 5.5
 neutrophiles
◦ growth optimum between pH 5.5 and pH 7
 alkaliphiles (alkalophiles)
◦ growth optimum between pH 8.5 and pH
11.5
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Temp affect living organisms in 2 ways:
- temp rise, chemical & enzymatic reaction
rise
- at very high temp, particular protein may
damage
 enzymes have optimal temperature at
which they function optimally
 high temperatures may inhibit enzyme
functioning
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organisms exhibit distinct
cardinal growth
temperatures
◦ minimal
◦ maximal
◦ optimal
psychrophiles – 0o C to 20o C
 psychrotrophs – 0o C to 35o C
 mesophiles – 20o C to 45o C
 thermophiles – 55o C to 85o C
 hyperthermophiles – 85o C to 113o C
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Microorganisms vary in their need
for/tolerance of O2.
 aerobe
◦ grows in presence of atmospheric
oxygen (O2) which is 20% O2
 Obligate (strict) aerobe – requires O2
 anaerobe
◦ grows in the absence of O2
 obligate anaerobe
◦ usually killed in presence of O2
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microaerophiles
◦ requires 2–10% O2
 facultative anaerobes
◦ do not require O2 but grow better in its
presence
 aerotolerant anaerobes
◦ grow with or without O2
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