Transcript Chapter 6

Chapter 6
Microbial Growth
Bacterial Cell Division
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New cells are formed by cell fission
Cells do not grow – they double their
cytoplasmic contents and membrane
They synthesize essential molecules
needed for their metabolic
processes
Binary Fission in Bacteria
Partitioning
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Prior to cell division, bacteria
copy their DNA( replicate
their DNA)
They then partition the DNA
by constructing a cell wall
between the two molecules
of DNA
This insures that the new cell
receives a copy of the
chromosome
The division or partitioning
of chromosomes is more
difficult in those organisms
that have more than one
chromosome
Prokaryote vs. Eukaryote
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Prokaryote cells do not go through
the cell cycle like eukaryote cells
They divide by fission
In some species there is some
linkage which forms tetrads,
sarcinae, and even staphylococci
Growth
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Increase in cellular constituents that may
result in:
– increase in cell number
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when microorganisms reproduce by budding or binary
fission
– increase in cell size
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coenocytic microorganisms have nuclear divisions that
are not accompanied by cell divisions. Fungi have a
syncytium and their nuclei are not separated.
Microbiologists usually study population
growth rather than growth of individual
cells
The Growth Curve
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Observed when microorganisms
are cultivated in batch culture
– culture incubated in a closed vessel
with a single batch of medium
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Usually plotted as logarithm of
cell number versus time
Usually has four distinct phases
population growth ceases
maximal rate of division
and population growth
no increase
Figure 6.1
decline in
population
size
Lag Phase
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Cell synthesizing new components
– to replenish spent materials
– to adapt to new medium or other
conditions
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varies in length
– in some cases can be very short or even
absent
Exponential Phase
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Also called log phase
Rate of growth is constant
Population is most uniform in terms
of chemical and physical properties
during this phase
cells are dividing and doubling in number at regular interval
Each individual
cell divides at a
slightly different
time
Curve rises
smoothly rather
than as discrete
steps
E. Coli Growth Curve
Balanced growth
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During log phase, cells exhibit
balanced growth
– cellular constituents manufactured at
constant rates relative to each other
Unbalanced growth
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Rates of synthesis of cell
components vary relative to each
other
Occurs under a variety of conditions
– change in nutrient levels
shift-up (poor medium to rich medium)
 shift-down (rich medium to poor medium)
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– change in environmental conditions
Stationary Phase
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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
Possible reasons for entry
into stationary phase
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nutrient limitation
limited oxygen availability
toxic waste accumulation
critical population density reached
Starvation responses
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morphological changes
– endospore formation
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decrease in size, protoplast
shrinkage, and nucleoid condensation
production of starvation proteins
long-term survival
increased virulence
Death Phase
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cells dying, usually at exponential
rate
death
– irreversible loss of ability to reproduce
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in some cases, death rate slows due
to accumulation of resistant cells
The Mathematics of
Growth
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Generation (doubling) time
– time required for the population to
double in size
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Mean growth rate constant
– number of generations per unit time
– usually expressed as generations per
hour
The Generation Time
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The generation time for most species is
between twenty minutes and 24 hours.
Some organisms take a longer time to go
through the lag phase
Some organisms due to their
characteristics like Mycobacterium
tuberculosis grow slowly due to the cell
wall
Synchronous Growth
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Cells doubling or dividing every 20
minutes
Measurement of
Microbial Growth
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Can measure changes in number of
cells in a population
Can measure changes in mass of
population
Measurement of Cell Numbers
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Direct cell counts
– counting chambers
– electronic counters
– on membrane filters
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Viable cell counts
– plating methods
– membrane filtration methods
Counting chambers
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easy,
inexpensive, and
quick
useful for
counting both
eucaryotes and
procaryotes
cannot
distinguish living
from dead cells
Figure 6.5
Electronic counters
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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
Electronic counters…
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cannot distinguish living from dead
cells
quick and easy to use
useful for large microorganisms and
blood cells, but not procaryotes
Direct counts on
membrane filters
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cells filtered through special
membrane that provides dark
background for observing cells
cells are stained with fluorescent
dyes
useful for counting bacteria
with certain dyes, can distinguish
living from dead cells
Plating methods
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plate dilutions of
population on suitable
Measure
solid medium
number of
viable cells

Population
count number of colonies
size is

expressed as
calculate number of cells
colony
in population
forming units
(CFU)
Spread Plate
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Samples are diluted by using 1 ml of broth
culture and 9 ml of sterile nutrient broth
Mix
Then 1 ml of the 1:10 ( first dilution) is
added to another 9ml of fresh nutrient
broth
Mix
Samples are diluted by using 1ml of broth
culture and 9 ml of sterile nutrient broth
Mix
Standard Dilutions
Spread plate
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A ml of each dilution is pipetted
with a plastic transfer pipet to the
center of an agar plate
A spreader( looks like a hockey
stick) is used to spread the cells
across the surface
This is designed to produce an even
distribution throughout
Colony Counter
Colony Counter
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To make an exact count of the colonies you place
the plate on a grid
You then illuminate the plate.
You count the colonies in the grid by going across
a horizontal row and then vertically to the next
row until you have covered the whole plate
The final count is multiplied x the dilution
factor. This number is the number of bacteria
that were in 1 ml of culture
It is assumed that each colony is equal to 1
original cell in the broth culture
Applications of this technique
commonly used in the
laboratory
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Determination of coliforms in the
environment( E. coli)
Determination of cells transformed
by genetic engineering
Determination of bacteria
contaminating soil in the
environment
Problems with colony
counts using plates
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There is error in this method
If the dilutions are homogeneous,
there can be errors
This may not capture all organisms in
a broth because some may not be
able to grow on the chosen media
Colony Counts
Pour Plates
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Add 1 ml of a serial
dilution to 9 ml of melted
and slightly warm nutrient
agar
Mix
Pour into a Petri dish and
allow it to harden
Colonies will develop both
in the media and on the
media
Cells may be damaged by
the hot agar in this
experiment
Plating methods…
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simple and sensitive
widely used for viable counts of
microorganisms in food, water, and
soil
inaccurate results obtained if cells
clump together
Most Probable Number
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Most probable number is used for environmental
samples
Trying to determine the presence of an organism
Use dilution factors as previously described
Use multiple tubes for dilutions
Check broth for cloudiness or turbidity( signs of
bacterial growth)
Use culture tubes containing sugars( lactose,
sucrose, glucose) These can be checked for the
presence of gas with a small tube on the interior
called a Durham tube.
See chart on page 149 for clarification
Membrane filtration
methods
Figure 6.6
especially useful for analyzing aquatic sample
Measurement of Cell Mass
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dry weight
– time consuming and not very sensitive
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quantity of a particular cell constituent
– protein, DNA, ATP, or chlorophyll
– useful if amount of substance in each cell is
constant

turbidometric measures (light scattering)
– quick, easy, and sensitive
more cells

more light
scattered

less light
detected
Figure 6.8
The Continuous Culture of
Microorganisms
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growth in an open system
– continual provision of nutrients
– continual removal of wastes
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maintains cells in log phase at a
constant biomass concentration
for extended periods
achieved using a continuous
culture system
The Chemostat
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rate of
incoming
medium = rate
of removal of
medium from
vessel
an essential
nutrient is in
limiting
quantities
Figure 6.9
Dilution rate and microbial
growth
dilution rate – rate at
which medium flows
through vessel
relative to vessel size
note: cell
density
maintained at
wide
range of
dilution
rates and
chemostat
operates best
at low dilution
rate
Figure 6.10
The Turbidostat
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regulates the flow rate of media
through vessel to maintain a
predetermined turbidity or cell
density
dilution rate varies
no limiting nutrient
turbidostat operates best at high
dilution rates
Importance of continuous
culture methods
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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
The Influence of
Environmental Factors on
Growth
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most organisms grow in fairly
moderate environmental conditions
extremophiles
– grow under harsh conditions that would
kill most other organisms
Solutes and Water
Activity
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water activity (aw)
– amount of water available to organisms
– reduced by interaction with solute
molecules (osmotic effect)
higher [solute]  lower aw
– reduced by adsorption to surfaces
(matric effect)
Osmotolerant organisms
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grow over wide ranges of water activity
many use compatible solutes to increase
their internal osmotic concentration
– solutes that are compatible with metabolism
and growth
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some have proteins and membranes that
require high solute concentrations for
stability and activity
Effects of NaCl on
microbial growth
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halophiles
– grow optimally at
>0.2 M
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extreme halophiles
– require >2 M
Figure 6.11
pH

negative logarithm
of the hydrogen
ion concentration
pH

acidophiles
– growth optimum between pH 0 and pH 5.5
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neutrophiles
– growth optimum between pH 5.5 and pH 7
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alkalophiles
– growth optimum between pH8.5 and pH 11.5
pH
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most acidophiles and alkalophiles maintain an
internal pH near neutrality
– some use proton/ion exchange mechanisms to do so
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some synthesize proteins that provide
protection
– e.g., acid-shock proteins
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many microorganisms change pH of their
habitat by producing acidic or basic waste
products
– most media contain buffers to prevent growth
inhibition
Temperature
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organisms
exhibit
distinct
cardinal
growth
temperatures
– minimal
– maximal
– optimal
Figure 6.13
Figure 6.14
Temperature and bacterial
growth
Adaptations of
thermophiles
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protein structure stabilized by a variety of
means
– more H bonds
– more proline
– chaperones
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histone-like proteins stabilize DNA
membrane stabilized by variety of means
– more saturated, more branched and higher
molecular weight lipids
– ether linkages (archaeal membranes)
Oxygen Concentration
need
oxygen
Figure 6.15
prefer
oxygen
ignore
oxygen
oxygen is
toxic
< 2 – 10%
oxygen
Basis of different oxygen
sensitivities
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oxygen easily reduced to toxic products
– superoxide radical
– hydrogen peroxide
– hydroxyl radical
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aerobes produce protective enzymes
– superoxide dismutase (SOD)
– catalase
Figure 6.14
Pressure
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barotolerant organisms
– adversely affected by increased
pressure, but not as severely as
nontolerant organisms
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barophilic organisms
– require or grow more rapidly in the
presence of increased pressure
Radiation
Radiation damage
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ionizing radiation
– x rays and gamma rays
– mutations  death
– disrupts chemical structure of many
molecules, including DNA
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damage may be repaired by DNA repair
mechanisms
Radiation damage…
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ultraviolet (UV) radiation
– mutations  death
– causes formation of thymine dimers in DNA
– DNA damage can be repaired by two
mechanisms
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photoreactivation – dimers split in presence of light
dark reactivation – dimers excised and replaced in
absence of light
Radiation damage…
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visible light
– at high intensities generates singlet
oxygen (1O2)
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powerful oxidizing agent
– carotenoid pigments
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protect many light-exposed microorganisms
from photooxidation
Microbial Growth in
Natural Environments
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microbial environments are complex,
constantly changing, and may expose
a microorganism to overlapping
gradients of nutrients and
environmental factors
Growth Limitation by
Environmental Factors
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Leibig’s law of the minimum
– total biomass of organism determined
by nutrient present at lowest
concentration
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Shelford’s law of tolerance
– above or below certain environmental
limits, a microorganism will not grow,
regardless of the nutrient supply
Responses to low nutrient
levels
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oligotrophic environments
morphological changes to increase
surface area and ability to absorb
nutrients
mechanisms to sequester certain
nutrients
Counting Viable but
Nonculturable Vegetative
Procaryotes
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stressed microorganisms can temporarily
lose ability to grow using normal
cultivation methods
microscopic and isotopic methods for
counting viable but nonculturable cells
have been developed
Quorum Sensing and
Microbial Populations
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quorum sensing
– microbial
communication and
cooperation
– involves secretion
and detection of
chemical signals
Figure 6.20
Processes sensitive to
quorum sensing: gramnegative bacteria
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bioluminescence (Vibrio fischeri)
synthesis and release of virulence
factors (Pseudomonas aeruginosa)
conjugation (Agrobacterium tumefaciens)
antibiotic production (Erwinia carotovora,
Pseudomonas aureofaciens)
biofilm production (P. aeruginosa)
Quorum sensing: grampositive bacteria
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often mediated by oligopeptide pheromone
processes impacted by quorum sensing:
– mating (Enterococcus faecalis)
– transformation competence (Streptococcus
pneumoniae)
– sporulation (Bacillus subtilis)
– production of virulence factors (Staphylococcus
aureus)
– development of aerial mycelia (Streptomyces
griseus)
– antibiotic production (S. griseus)
The Lux Gene in Vibrio
Fischeri
Requirements for Nitrogen
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Nitrogen is required for the synthesis of amino acids that
compose the structure of proteins, purines and pyrimidines
the bases of both DNA and RNA, and for other derivative
molecules such as glucosamine.
Many microorganisms can use the nitrogen directly from
amino acids. The amino group ( NH2) is derived from
ammonia through the action of enzymes such as glutamate
dehydrogenase.
Most photoautotrophs and many nonphotosynthetic
microorganisms reduce nitrate to ammonia and assimilate
nitrogen through nitrate reduction. A variety of bacteria
are involved in the nitrogen cycle such as Rhizobium which
is able to use atmospheric nitrogen and convert it to
ammonia. ( Found on the roots of legumes like soy beans
and clover) These compounds are vital for the Nitrogen
cycle and the incorporation of nitrogen into plants to make
nitrogen comounds.
Phosphorous
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
Phosphorous is present in phospholipids(
membranes), Nucleic acids( DNA and
RNA), coenzymes, ATP, some proteins,
and other key cellular components.
Inorganic phosphorous is derived from
the environment in the form of
phosphates. Some microbes such as E.
coli can use organophosphates such as
hexose – 6-phosphates .
Mixotrophy
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Chemical energy – source organic
Inorganic H/e- donor
Organic carbon source
Requirements for Nitrogen,
Phosphorus, and Sulfur
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Needed for synthesis of important
molecules (e.g., amino acids, nucleic acids)
Nitrogen supplied in numerous ways
Phosphorus usually supplied as inorganic
phosphate
Sulfur usually supplied as sulfate via
assimilatory sulfate reduction
Sources of nitrogen
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organic molecules
ammonia
nitrate via assimilatory nitrate
reduction
nitrogen gas via nitrogen fixation
Growth Factors
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organic compounds
essential cell components (or their
precursors) that the cell cannot
synthesize
must be supplied by environment if
cell is to survive and reproduce
Classes of growth factors

amino acids
– needed for protein synthesis

purines and pyrimidines
– needed for nucleic acid synthesis

vitamins
– function as enzyme cofactors
Amino acids
Proteins
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Bases of nucleic acids
Adenine and guanine
are purines
Cytosine, thymine,
and uracil are
pyrimidines
Also found in energy
triphosphates( ATP
and GTP)
Practical importance of
growth factors
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
development of quantitative growthresponse assays for measuring
concentrations of growth factors in
a preparation
industrial production of growth
factors by microorganisms
Uptake of Nutrients
by the Cell
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
Some nutrients enter by passive
diffusion
Most nutrients enter by:
– facilitated diffusion
– active transport
– group translocation
Passive Diffusion


molecules move from region of
higher concentration to one of lower
concentration because of random
thermal agitation
H2O, O2 and CO2 often move across
membranes this way
Active Transport

energy-dependent process
– ATP or proton motive force used



moves molecules against the
gradient
concentrates molecules inside cell
involves carrier proteins
(permeases)
– carrier saturation effect is observed
Transporters

“Molecular Properties of Bacterial Multidrug
Transporters”
–
Monique Putnam, Hendrik van Veen, and Wil
Konings – PubMed Central. Full Text available .
Microbiol Mol Biol Review. 2000 December; 64 (4): 672–
693
ABC transporters
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
ATP-binding
cassette
transporters
observed in
bacteria,
archaea, and
eucaryotes
Figure 5.3
antiport
Figure 5.4
symport
Group Translocation


molecules are
modified as
they are
transported
across the
membrane
energydependent
process
Figure 5.5
Fe uptake in pathogens
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The ability of pathogens to obtain iron
from transferrins, ferritin, hemoglobin,
and other iron-containing proteins of
their host is central to whether they live
or die
Some invading bacteria respond by
producing specific iron chelators siderophores that remove the iron from
the host sources. Other bacteria rely on
direct contact with host iron proteins,
either abstracting the iron at their
surface or, as with heme, taking it up into
Iron and signalling


Iron is also used by pathogenic bacteria
as a signal molecule for the regulation of
virulence gene expression. This sensory
system is based on the marked
differences in free iron concentrations
between the environment and intestinal
lumen (high) and host tissues (low)
Listeria Pathogenesis and Molecular Virulence Determinants
José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5
Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen
Wehland,6 and Jürgen Kreft3
Pathogens and Iron uptake

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Burkholderia cepacia
Campylobacter jejuni
Pseudomonas aeruginosa
E. coli
Listeria monocytogenes
Iron Uptake

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ferric iron is very
insoluble so uptake is
difficult
microorganisms use
siderophores to aid
uptake
siderophore complexes
with ferric ion
complex is then
transported into cell
Figure 5.6
Listeriosis



One involves the direct transport of
ferric citrate to the bacterial cell
Another system involves an
extracellular ferric iron reductase,
which uses siderophores
The third system may involve a
bacterial cell surface-located
transferrin-binding protein
Iron bacteria in the
environment


There are several non-disease producing
bacteria which grow and multiply in water
and use dissolved iron as part of their
metabolism. They oxidize iron into its
insoluble ferric state and deposit it in the
slimy gelatinous material which surrounds
their cells.
These filamentous bacteria grow in
stringy clumps and are found in most ironbearing surface waters. They have been
known to proliferate in waters containing
iron as low as 0.1 mg/l.
Culture Media


preparations devised to support the
growth (reproduction) of
microorganisms
can be liquid or solid
– solid media are usually solidified with
agar

important to study of microorganisms
Synthetic or Defined
Media

all components
and their
concentrations
are known
Complex Media

contain some
ingredients of
unknown
composition
and/or
concentration
Some media components

peptones
– protein hydrolysates prepared by partial
digestion of various protein sources

extracts
– aqueous extracts, usually of beef or yeast

agar
– sulfated polysaccharide used to solidify liquid
media
Types of Media

general purpose media
– support the growth of many microorganisms
– e.g., tryptic soy agar

enriched media
– general purpose media supplemented by blood
or other special nutrients
– e.g., blood agar
Types of media…

Selective media
– Favor the growth of some
microorganisms and inhibit growth of
others
– MacConkey agar
selects for gram-negative bacteria
 Inhibits the growth of gram-positive
bacteria

Beta Hemolysis
Types of media…

Differential media
– Distinguish between different groups of
microorganisms based on their biological
characteristics
– Blood agar

hemolytic versus nonhemolytic bacteria
– MacConkey agar

lactose fermenters versus nonfermenters
Selective and
differential media
Selects for Gram –
Differentiates between
bacteria based upon
fermentation of lactose(
color change)
Organism
Salt Tolerance
Mannitol Fermentation
1. S. aureus
Positive - growth
Positive (yellow)
2. S. epidermidis
Positive*- growth
Negative( color does not change) – no fermentation of mannitol with
production of acid
3. M. luteus
Negative
N/A**
http://www.austin.cc.tx.us/microbugz/20msa.html
Web References on Media
http://www.jlindquist.net/generalmicro/102diff.html - General
Reference
http://medic.med.uth.tmc.edu/path/macconk.htm - MacConkey
Agar
http://www.indstate.edu/thcme/micro/hemolys.html - Blood
Agar
Spread-plate technique
1. dispense cells onto
medium in petri dish
Figure 5.7
4. spread cells
across surface
2. - 3. sterilize spreader
Streak plate technique
inoculating
loop
Figure 5.8
Isolation of Pure Cultures

Pure culture
– population of cells arising from a single
cell

Spread plate, streak plate, and pour
plate are techniques used to isolate
pure cultures
Colony Morphology and Growth

individual
species form
characteristic
colonies
Figure 5.10b
Terms
1. Colony shape and size: round, irregular, punctiform
(tiny)
2. Margin (edge): entire (smooth), undulate (wavy), lobate
(lobed)
3. Elevation: convex, umbonate, flat, raised
4. Color: color or pigment, plus opaque, translucent, shiny
or dull
5. Texture: moist, mucoid, dry (or rough).
Figure 5.10a
Colony growth

Most rapid at edge of colony
– oxygen and nutrients are more available
at edge


Slowest at center of colony
In nature, many microorganisms
form biofilms on surfaces