M I C R O B I O L O G Y
a n i n t r o d u c t i o n
ninth edition TORTORA⏐FUNKE⏐CASE
6
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PowerPoint®Lecture Slide Presentation prepared by Christine L. Case
6
Microbial
Growth
Microbial growth is the increase in number of cells, not cell size
Microbial Growth
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The Requirements for Growth:
Physical Requirements
Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
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Temperature
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Psychrotrophs
Grow between 0°C and 20-30°C
Cause food spoilage
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Psychrotrophs
The Requirements for Growth:
Physical Requirements
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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The Requirements for Growth:
Physical Requirements
Osmotic pressure
Hypertonic environments, increase salt or sugar, cause plasmolysis
Extreme or obligate halophiles require high osmotic
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pressure
Facultative halophiles tolerate high osmotic pressure
The Requirements for Growth:
Physical Requirements
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The Requirements for Growth:
Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2
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The Requirements for Growth:
Chemical Requirements
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+or NO3–
A few bacteria use N2in nitrogen fixation
S lf
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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
The Requirements for Growth:
Chemical Requirements
Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors
The Requirements for Growth:
Chemical Requirements
Oxygen (O2)
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Toxic Forms of Oxygen
Singlet oxygen: O2boosted to a higher-energy state
Superoxide free radicals: O2–
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Peroxide anion: O22–
Hydroxyl radical (OH•)
The Requirements for Growth:
Chemical Requirements
Organic growth factors
Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines
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Culture Media
Culture medium: Nutrients prepared for microbial growth
Sterile: No living microbes
Inoculum: Introduction of microbes into medium
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Culture: Microbes growing in/on culture medium
Agar
Complex polysaccharide
Used as solidifying agent for culture media in Petri plates, slants, and deeps
Generally not metabolized by microbes
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Liquefies at 100°C
Solidifies ~40°C
Culture Media
Chemically defined media: Exact chemical composition is known
Complex media: Extracts and digests of yeasts, meat, or plants
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Nutrient broth
Culture Media
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Growth in Continuous Culture
A “continuous culture” is an open system in which
fresh media is continuously added to the culture at
a constant rate, and old broth is removed at the
same rate.
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This method is accomplished in a device called a
chemostat.
Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as
the nutrient feed is maintained.
Basic Chem ost at Syst em
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Our Ch em ost at Syst em
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Anaerobic Culture Methods
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2
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Anaerobic Culture Methods
Anaerobic jar
Anaerobic Culture Methods
Anaerobic chamber
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Capnophiles Require High CO
2 Candle jar
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CO2-packet
Figure 6.7
Selective Media
Suppress unwanted microbes and
encourage desired
microbes.
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Selective Media
Inhibits the growth of some bacteria while selecting for the growth of others
Example:
Brilliant Green Agar
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dyes inhibit the growth of Gram (+) bacteria
selects for Gram (-) bacteria
Most G.I. Tract infections are caused by Gram (-) bacteria
Selective Media
EMB (Eosin Methylene Blue)
dyes inhibit Gram (+) bacteria
selects for Gram (-) bacteria
G.I. Tract infections caused by Gram (-)
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bacteria
Differential Media
Make it easy to distinguish colonies of different microbes.
Some media are both selective and
differential
Mannitol salt agaris both selective and differential
Selective:
Staphylococcus aereuscan grow on mannitol salt agar that has a high concentration of salt; the growth of other organisms will be inhibited
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Differential:
Staphylococcus aureusferments mannitol and the medium will change color
Other organisms that grow on high salt will grow on mannitol salt agar but may not ferment mannitol; the media will not change colors
Selective and Differential Media
Mannitol Salt Agar
used to identify Staphylococcus aureus
M i l S l A
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Mannitol Salt Agar
High salt conc. (7.5%) inhibits most bacteria
sugar Mannitol
pH Indicator (Turns Yellow when acid)
Selective and Differential Media
MacConkey’s Agar
used to identify Salmonella
MacConkey’s Agar
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Bile salts and crystal violet (inhibits Gram (+) bacteria)
lactose
pH Indicator
Many Gram (-) enteric non-pathogenic bacteria can ferment lactose, Salmonella can not
Enrichment Media
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
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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
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).
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Streak Plate
Preserving Bacteria Cultures
Deep-freezing: –50°to –95°C
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum
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Reproduction in Prokaryotes
Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
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Binary Fission
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If 100 cells growing for 5 hours produced 1,720,320 cells:
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PLAY Animation: Bacterial Growth
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1. Lag Phase
Bacteria are first introduced into an environment or media
Bacteria are “checking out” their surroundings
cells are very active metabolically
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# of cells changes very little
1 hour to several days
2. Log Phase
Rapid cell growth (exponential growth)
population doublesevery generation
microbes are sensitive to adverse conditions
antibiotics
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anti-microbial agents
3. Stationary Phase
Death rate = rate of reproduction
cells begin to encounter environmental stress
lack of nutrients
lack of water
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not enough space
metabolic wastes
oxygen
pH
Endospores would form now
4. Death Phase
Death rate > rate of reproduction
Due to limiting factors in the environment
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Measuring Microbial Growth
Direct methods
Plate counts
Filtration
MPN
Indirect methods
Turbidity
Metabolic activity
Dry weight
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Direct microscopic count
Dry weight
Direct Measurements of Microbial Growth
Plate counts: Perform serial dilutions of a sample
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Plate Count
Inoculate Petri plates from serial
dilutions
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Plate Count
After incubation, count colonies on plates that have 25-250 colonies (CFUs)
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Direct Measurements of Microbial Growth
Filtration
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Direct Measurements of Microbial Growth
Multiple tube MPN test.
Count positive tubes and
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compare to
statistical
MPN table.
Figure 6.18b
Direct Measurements of Microbial Growth
Direct microscopic count
Direct Measurements of Microbial Growth
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Estimating Bacterial Numbers
by Indirect Methods
Turbidity