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Physical requirements for growth
• Prefixes and suffixes:
• Bacteria are highly diverse in the types of
conditions they can grow in.
– Optimal or required conditions implied by “phile” meaning “love”
• Some bacteria prefer other conditions,
but can tolerate extremes
– Suffix “-tolerant”
• Note the difference!
http://www.kodak.com/global/images/en/health/filmImaging/thermometer.gif
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When growing microbes..
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• The physical/chemical conditions that are most
important
– Presence or absence of oxygen
– Temperature range
– pH range
– Water activity (how “wet”)
• Note that by changing the conditions to make them
unfavorable we can prevent bacterial growth.
Oxygen: friend or foe?
• Early atmosphere of Earth had none
– First created by cyanobacteria using photosynthesis
– Iron everywhere rusted, then collected in atmosphere
• Strong oxidizing agent
• Reacts with certain organic molecules, produces
free radicals and strong oxidizers :
– Singlet oxygen, H2O2(peroxide), O3- (superoxide), and
hydroxyl (OH-) radical.
– Free radicals are highly reactive chemicals that damage
proteins, nucleic acids, and other cell molecules.
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Protections of bacteria against oxygen
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– Bacteria possess protective enzymes, catalase and
superoxide dismutase.
– Catalase breaks down hydrogen peroxide into water and
oxygen gas.
– Superoxide dismutase breaks superoxide down into
peroxide and oxygen gas.
– Anaerobes missing one or both; slow or no growth in
the presence of oxygen.
Fe3+ -SOD + O2- → Fe2+ -SOD + O2
Fe2+ -SOD + O2- + 2H+ → Fe 3+ -SOD + H2O2
Relation to Oxygen
• Aerobes: use oxygen in
metabolism; obligate.
A: aerobe
B: microaerophile
• Microaerophiles: require oxygen
(also obligate), but in small
amounts.
• Anaerobes: grow without oxygen;
SEE NEXT
•Capnophiles: require larger amounts of carbon dioxide than are found normally in air.
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Anaerobes grow without O2
• Classifications vary, but our
definitions:
– Obligate (strict) anaerobes:
killed or inhibited by oxygen.
– Aerotolerant anaerobes: do
not use oxygen, but not killed
by it.
– Facultative anaerobes: can
grow with or without oxygen
C: could be facultative or
aerotolerant.
D: strict anaerobe
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Effect of temperature
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• Low temperature
– Enzymatic reactions too slow; enzymes too stiff
– Lipid membranes no longer fluid
• High temperature
– Enzymes denature, lose shape and stop functioning
– Lipid membranes get too fluid, leak
– DNA denatures
• As temperature increases, reactions and growth
rate speed up; at max, critical enzymes
denature.
Bacteria and temperature
• Bacteria have temperature ranges (grow between 2
temperature extremes), and an optimal growth
temperature. Both are used to classify bacteria.
• As temperature increases, so do metabolic rates.
• At high end of range, critical enzymes begin to
denature, work slower. Growth rate drops off rapidly
with small increase in temperature.
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Classification of bacteria based on
temperature
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Terms related to temperature
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• Special cases:
– Psychrotrophs: bacteria that grow at “normal”
(mesophilic) temperatures (e.g. room temperature”
but can also grow in the refrigerator; responsible for
food spoilage.
– Thermoduric: more to do with survival than growth;
bacteria that can withstand brief heat treatments.
pH Effects
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• pH = -log[H+]
• Lowest = 0 (very acid); highest = 14 (very
basic) Neutral is pH 7.
• Acidophiles/acidotolerant grow at low pH
• Alkalophiles/alkalotolerant grow at high pH
• Most bacteria prefer a neutral pH
– Many grow well from pH 6 to 8
• Some bacteria create their preferred conditions
– Lactobacillus creates low pH environment in vagina
Low water activity:
halophiles, osmophiles, and xerotolerant
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• Water is critical for life; remove some, and things can’t
grow. (food preservation: jerky, etc.)
• Halophiles/halotolerant: relationship to high salt.
– Marine bacteria; archaea and really high salt.
• Osmophiles: can stand hypertonic environments
whether salt, sugar, or other dissolved solutes
– Fungi very good at this; grandma’s wax over jelly.
• Xerotolerant: dry. Subject to desiccation. Fungi best
– Bread, dry rot of wood
– Survival of bacterial endospores.
Bacterial growth defined
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• Since individual cells double in size, then divide into
two, the meaningful increase is in the population size.
• Binary fission: cell divides into two cells. No nucleus,
so no mitosis.
• Cells do not always fully detach; produce pairs,
clusters, chains, tetrads, sarcina, etc.
• “GROWTH” = increase in number of bacteria
Mathematics of bacterial growth
• Because bacteria double in
number at regular intervals,
they grow exponentially:
• N = N0 x 2n where N is the
number of cells after n
number of doublings and N0
is the starting number of
cells.
• Thus, a graph of the Log of
the number of bacteria vs.
time is a straight line.
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The Bacterial Growth Curve
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log (# of bacteria)
• Bacteria provided with an abundant supply of
nutrients will increase in number exponentially, but
eventually run out of nutrients or poison themselves
with waste products.
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2
1
tim e
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1. Lag phase
2. Exponential or
Log phase
3. Stationary
phase
4. Decline or
Death
phase.
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• Lag phase: growth lags; cells
are acclimating to the medium,
creating ribosomes prior to
rapid growth.
• Log phase: cells doubling at
regular intervals; linear graph
when x-axis is logarithmic.
log (# of bacteria)
Growth curve (continued)
time
•Stationary phase: no net increase in cell numbers, some
divide, some die. Cells preparing for survival.
•Decline phase: highly variable, depends on type of
bacteria and conditions. Death may be slow and
exponential.
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More about Growth
• The Growth curve is true under ideal conditions; in
reality, bacteria are subject to starvation, competition,
and rapidly changing conditions.
• Generation time: the length of time it takes for the
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population to double.
•Growth of bacteria is nonsynchronous,
not every bacterium is dividing at the
same time.
•Instead of stepwise curve, smooth
curve
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Exponential growth
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• “Balanced growth”
– Numbers of bacteria are doubling at regular
intervals.
– All components of bacteria are increasing in amount
at the same rate
• 2x as many bacteria = 2x as much protein, 2x as
much peptidgolycan, 2x as much LPS, etc.
– During exponential growth, bacteria are not limited
for any nutrients, i.e. they are not short of anything.
Measurement of cell numbers
• Direct methods: cells
actually counted.
– Petroff-Hausser
counting chamber
(right), 3D grid. Count
the cells, multiply by a
conversion factor.
– Dry a drop of cells of
known volume, stain,
then count.
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Coulter Counter
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Coulter-counter: singlefile cells detected by
change in electric
current.
Counting cells with plates
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• Viable plate count
– Relies on bacteria being alive,
multiplying and forming
colonies.
– Spread plate: sample is spread
on surface of agar.
– Pour plate: sample is mixed
with melted agar; colonies form
on surface and within agar.
– Colonies counted with a colony
counter.
biology.clc.uc.edu/.../Meat_Milk/ Pour_Plate.htm
Filtration and plate counting
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•Membrane filters are very thin
with a defined pore size, e.g.
0.45 µm.
•Bacteria from a dilute sample are
collected on a filter; filter placed
on agar plate, colonies counted.
•Used when concentration of
bacteria is low.
http://dl.clackamas.cc.or.us/wqt111/coliform-8.jpg
http://www.who.int/docstore/water_sanitation_health/labman
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Spectrophotometry
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• Bacteria scatter light, making a turbid (cloudy)
suspension.
• Turbidity is usually read on the Absorbance scale
– Not really absorbance, but Optical Density (OD)
• More bacteria, greater the turbidity (measured as OD)
Based on www.umr.edu/~gbert/ color/spec/Aspec.html
More about Spectrophotometry
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– Does NOT provide an actual number unless a
calibration curve (# of bacteria vs. O.D.) is created.
• Indirect counting method
– Quick and convenient, shows relative change in the
number of bacteria, useful for determining growth
(increase in numbers).
– Does NOT distinguish between live and dead cells.
To create a calibration curve, best to plot OD vs.
number of cells determined with microscope (not
plate count).
Biomass:
• Measure the total mass of cells or amount of any
component such as protein, PS, DNA, KDO.
• Especially when cells are doubling,
the amounts of all the components
of a cell are increasing at the same
rate, so any could be measured.
– Not so in stationary phase.
In this example, total biomass
increases exponentially over time.
http://www.pubmedcentral.nih.gov/pagerende
r.fcgi?artid=242188&pageindex=10#page
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