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Copyright © 2013 Pearson Education, Inc.Lectures prepared by Christine L. Case
Chapter 6
Microbial Growth
© 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case
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Microbial Growth
Increase in number of cells, not cell size
Populations
Colonies
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The Requirements for Growth
Physical requirements
Temperature
pH
Osmotic pressure
Chemical requirements
Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factor
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Physical Requirements
Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
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Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature.
PsychrophilesPsychrotrophs
Mesophiles
Thermophiles
Hyperthermophiles
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Affect enzyme reaction
Denature enzyme, carrier & protein
Disrupt membrane
1. psychrophile: 0-20°C, opt. 15°C
2. psychrotroph: 0-35°C, opt. 20-30°C
3. mesophile: (15-20°C)-45°C, opt. 20-45°C
most MO in this group
4. thermophile: bacteria,min 45°C, opt. 55-65°C
5.hyperthermophile: archaea, opt.80°C or greater
have much more heat-stable en. & protein
membrane lipid more saturated
Temperature vs. Microorganisms
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Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent.
Water is being emitted through the ocean floor at temperatures above 100°C.
Chemoautotrophic bacteria in
deep-sea water use chemical
energy from H2S as a source of
energy to fix CO2.
Hydrothermal Vent-
•Next frontier for new drugs
•Produce alternative fuels, H2
and butanol
•Provide heat-resistant
enzymes, eg. DNA polymerase
used for PCR
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Figure 6.2 Food preservation temperatures.
Temperatures in this range destroy most microbes,
although lower temperatures take more time.
Very slow bacterial growth.
Rapid growth of bacteria; some may produce toxins.
Many bacteria survive; some may grow.
Refrigerator temperatures; may allow slow growth
of spoilage bacteria, very few pathogens.
No significant growth below freezing.
Danger zone
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Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage.
Refrigerator air
5 cm (2′′) deep
15 cm (6′′) deep
Approximate temperature
range at which Bacillus cereus
multiplies in rice
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pH
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
Acidophiles grow in acidic environments
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Osmotic Pressure
Hypertonic environments, or an increase in salt or
sugar, cause plasmolysis
Extreme or obligate halophiles require high
osmotic pressure
Facultative halophiles tolerate high osmotic
pressure
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pH
Most bacteria grow well in neutral pH (pH 6.5 - 7.5)
(neutrophile). Few are acidophiles, eg. One in the
drainage water from coal mines oxidezes sulfur to form
sulfuric acid and can survive at pH 1.0.
Molds and yeasts: pH 1-11, opt. pH 5- 6
Acidophiles: pH 1-5.5
Alkalophile: pH 8.5-11.5
The Requirements for Growth: Physical Requirements
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Figure 6.4 Plasmolysis.
Plasma
membraneCell wall
Cytoplasm
H2O
NaCl 10%
Cytoplasm
Plasma
membrane
Cell in isotonic solution. Plasmolyzed cell in hypertonic
solution.
NaCl 0.85%
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Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2
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Chemical Requirements
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3
–
A few bacteria use N2 in nitrogen fixation
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Chemical Requirements
Sulfur
In amino acids, thiamine, and biotin
Most bacteria decompose proteins
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus
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Chemical Requirements
Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors
Na, K, Mg, Ca, S, P, in mg/ml scale
Fe, Cu, Mo, Zn in ng/ml scale
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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria
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Singlet oxygen: 1O2− boosted to a higher-energy
state
Superoxide free radicals: O2
Peroxide anion: O22–
Hydroxyl radical (OH•)
Toxic Oxygen
Superoxide dismutaseO2 + O2 + 2 H+ H2O2 + O2
Catalase2 H2O2 2 H2O + O2
PeroxidaseH2O2 + 2 H+ 2 H2O
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Both obligate aerobe and facultative anaerobe: have SOD
and catalase
Obligate anaerobe: without SOD & catalase, O2 sensitive
Aerotolerant anaerobe: not use O2 for growth, tolerate O2,
have SOD or equivalent enzyme to neutralize O2 toxicity,
lactobacilli
Microaerophile: require less O2, sensitive to superoxide anion
and peroxide produced under O2-rich condition
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Organic Growth Factors
Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines
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Biofilms
Microbial communities
Form slime or
hydrogels
Bacteria attracted by
chemicals via quorum
sensing
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Biofilms
Share nutrients
Sheltered from harmful factors
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Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.
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Biofilms
Patients with indwelling catheters (留置導尿管)
received contaminated heparin
Bacterial numbers in contaminated heparin were too
low to cause infection
84–421 days after exposure, patients developed
infections
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Culture Media
Culture medium: nutrients prepared for microbial
growth
Sterile: no living microbes
Inoculum: introduction of microbes into medium
Culture: microbes growing in/on culture medium
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Agar
Complex polysaccharide
Used as solidifying agent for culture media in Petri
plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 100°C
Solidifies at ~40°C
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Culture Media
Chemically defined media: exact chemical
composition is known
Complex media: extracts and digests of yeasts,
meat, or plants
Nutrient broth
Nutrient agar
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Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia
coli
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Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides
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Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria
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Anaerobic Culture Methods
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine
O2, oxyrase is a respiratory enzyme that can reduce O2 to
water
OxyPlate: plate with oxyrase, used in clinical lab.
Heated to drive off O2
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Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Lid with
O-ring gasket
Envelope containing
sodium bicarbonate
and sodium
borohydride
Anaerobic indicator
(methylene blue)
Petri plates
Clamp with
clamp screw
Palladium
catalyst pellets
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Figure 6.7 An anaerobic chamber.
Arm
ports
Air
lock
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Capnophiles
Microbes that require high CO2 conditions
CO2 packet
Candle jar
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Candle jar
Capnophiles require high CO2
Figure 6.7
•CO2-packet
Low O2, high CO25% O2, 10% CO2
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BSL-1: no special precautions
BSL-2: lab coat, gloves, eye protection
BSL-3: biosafety cabinets to prevent airborne
transmission
BSL-4: sealed, negative pressure Exhaust air is filtered twice
Biosafety Levels
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Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.
© 2013 Pearson Education, Inc. Figure 6.10
Selective Media
Suppress unwanted
microbes and encourage
desired microbes
Eg. Bismuth sulfite agar for
Salmonella typhi, Bismuth
sulfite: inhibit G(+) and
most G(-) intestinal bact.
Brilliant green agar for G(-)
Salmonella
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Eosine methylene
blue (EMB) agar:
E. coli: black-center
colony, surrounded
by metallic green
sheen
Enterbacter aerogenes:
Dark-center colony
Differential media : Make it easy to distinguish
colonies of different microbes.
Figure 6.9b, c
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Figure 6.9 Blood agar, a differential medium containing red blood cells.
Bacterial
colonies
Hemolysis
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Enrichment Culture
Encourages growth of desired microbe
Assume a soil sample contains a few
phenol-degrading bacteria and thousands of
other bacteria
Inoculate phenol-containing culture medium with the soil,
and incubate
Transfer 1 ml to another flask of the phenol medium, and
incubate
Transfer 1 ml to another flask of the phenol medium, and
incubate
Only phenol-metabolizing bacteria will be growing
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Obtaining Pure Cultures
A pure culture contains only one species or strain
A colony is a population of cells arising from a
single cell or spore or from a group of attached cells
A colony is often called a colony-forming unit (CFU)
The streak plate method is used to isolate pure
cultures
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Figure 6.11 The streak plate method for isolating pure bacterial cultures.
Colonies
1
2
3
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Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C
Lyophilization (freeze-drying): frozen
(–54° to –72°C) and dehydrated in a vacuum
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Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
ANIMATION Bacterial Growth: Overview
Reproduction in Prokaryotes
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Figure 6.12a Binary fission in bacteria.
Cell elongates and
DNA is replicated.
Cell wall and plasma
membrane begin to constrict.
Cross-wall forms,
completely separating
the two DNA copies.
Cells separate.
Cell wall
Plasma
membrane
DNA
(nucleoid)
(a) A diagram of the sequence of cell division
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Figure 6.12b Binary fission in bacteria.
(b) A thin section of a cell of Bacillus licheniformis
starting to divide
Cell wallDNA (nucleoid)
Partially formed cross-wall
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Figure 6.13a Cell division.
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Figure 6.13b Cell division.
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If 100 cells growing for 5 hours produced
1,720,320 cells:
ANIMATION Binary Fission
Generation Time
Number of generations =Log number of cells (end) − Log number of cells (beginning)
0.301
Generation time =60 min × hours
Number of generations= 21 minutes/generation
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Nt = No x 2n
Log Nt = Log No +n Log 2
n Log 2 = Log Nt – Log No
n =(Log Nt-Log No)/Log 2
n= number of generation =incubation time(t) /generation time (tg)
No : initial number
Nt : final number after t time incubation
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The Growth Curve
observed when microorganisms are
cultivated in batch culture
culture incubated in a closed vessel with
a single batch of medium
usually plotted as logarithm of cell
number versus time
usually has four distinct phases
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Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line)
and arithmetically (solid line).
Log10 = 1.51
Log10 = 3.01
Log10 = 4.52
Log10 = 6.02
(1,048,576)
Generations
Lo
g1
0 o
f n
um
be
r o
f cell
s
Nu
mb
er
of
cell
s
(32) (1024)
(32,768)
(65,536)
(131,072)
(262,144)
(524,288)
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Lag PhaseIntense activity
preparing for
population growth,
but no increase in
population.
Log PhaseLogarithmic, or
exponential,
increase in
population.
Stationary PhasePeriod of equilibrium;
microbial deaths
balance production of
new cells.
Death PhasePopulation Is
decreasing at a
logarithmic rate.
The logarithmic growth
in the log phase is due to
reproduction by binary
fission (bacteria) or
mitosis (yeast).
Figure 6.15 Understanding the Bacterial Growth Curve.
Staphylococcus spp.
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Bacterial growth phase
Lag phase: little or on cell division
prepare to synthesize enzymes and molecules for adaptation to a new environment
Log phase: cell number increases logarithmically
cells are most active metabolically, very sensitive to adverse conditions
Stationary phase: cell growth and cell death balance the total number
exhaustion of nutrients, accumulation of waste, harmful pH
Death phase: numbers of death exceeds the number of new formed
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Lag Phase
cell synthesizing new components
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
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Effect of nutrient concentration on growth
Figure 6.7
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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
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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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Death Phase
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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Measurement of Cell Numbers
direct cell counts
counting chambers
electronic counters
on membrane filters
viable cell counts
plating methods
membrane filtration methods
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Plating methods…
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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Figure 6.16 Serial dilutions and plate counts.
Original
inoculum
1 ml 1 ml 1 ml 1 ml 1 ml
9 m broth
in each tube
Dilutions 1:10 1:100 1:1000 1:10,000 1:100,000
1 ml 1 ml 1 ml 1 ml 1 ml
1:10 1:100 1:1000 1:10,000 1:100,000(10-1) (10-2) (10-3) (10-4) (10-5)
Plating
Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml
(For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.)
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After incubation, count colonies on plates that have
25–250 colonies (CFUs)
Plate Counts
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Figure 6.17 Methods of preparing plates for plate counts.
The pour plate method The spread plate method
Inoculate
empty plate.
Add melted
nutrient
agar.
Swirl to
mix.
Colonies
grow on
and in
solidified
medium.
1.0 or 0.1 ml 0.1 ml
Bacterial
dilution
Inoculate plate
containing
solid medium.
Spread inoculum
over surface
evenly.
Colonies grow
only on surface
of medium.
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Another Viable Count Method - Membrane filtration
especially useful for analyzing aquatic samples
Figure 6.13
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Figure 6.18 Counting bacteria by filtration.
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Figure 6.14
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Multiple tube MPN test
Count positive tubes
Most Probable Number
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Figure 6.19a The most probable number (MPN) method.
Volume of
Inoculum for
Each Set of
Five Tubes
(a) Most probable number (MPN) dilution series.
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Compare with a statistical table
Most Probable Number
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Figure 6.19b The most probable number (MPN) method.
(b) MPN table.
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Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter.
Grid with 25 large squares
Cover glass
Slide
Bacterial suspension is added here and fills the
shallow volume over the squares by capillary
action.
Bacterial
suspension
Cover glass
Slide
Cross section of a cell counter.
The depth under the cover glass and the area
of the squares are known, so the volume of the
bacterial suspension over the squares can be
calculated (depth × area).
Microscopic count: All cells in
several large squares are
counted, and the numbers are
averaged. The large square shown
here has 14 bacterial cells.
The volume of fluid over the
large square is 1/1,250,000
of a milliliter. If it contains 14 cells,
as shown here, then
there are 14 × 1,250,000 =
17,500,000 cells in a milliliter.
Location of squares
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Direct Microscopic Count
Number of bacteria/ml =Number of cells counted
Volume of area counted
14
8 × 10−7= 17,500,000
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Figure 6.21 Turbidity estimation of bacterial numbers.
Light source
Light
Blank
Spectrophotometer
Light-sensitive
detectorScattered light
that does not
reach detector
Bacterial suspension
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Measuring Microbial Growth
Direct Methods
Plate counts
Filtration
MPN
Direct microscopic count
Indirect Methods
Turbidity
Metabolic activity
Dry weight