Transcript pure culture

Microbial Growth
The Requirements for Growth
• Physical requirements
– Temperature
– pH
– Osmotic pressure
• Chemical requirements
–
–
–
–
–
Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factor
The Requirements for Growth:
Physical Requirements
• Temperature
– Minimum growth temperature
– Optimum growth temperature
– Maximum growth temperature
Temperature
Figure 6.1
Psychrotrophs
• Grow between 0°C and 20-30°C
• Cause food spoilage
Psychrotrophs
Figure 6.2
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
The Requirements for Growth:
Physical Requirements
• Osmotic Pressure
– Hypertonic environments, increase salt or
sugar, cause plasmolysis
– Extreme or obligate halophiles require high
osmotic pressure
– Facultative halophiles tolerate high osmotic
pressure
The Requirements for Growth:
Physical Requirements
Figure 6.4
The Requirements for Growth:
Chemical Requirements
• Carbon
– Structural organic molecules, energy source
– Chemoheterotrophs use organic carbon sources
– Autotrophs use CO2
• Nitrogen
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–
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In amino acids, proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3
A few bacteria use N2 in nitrogen fixation
• Sulfur
– In amino acids, thiamine, biotin
– Most bacteria decompose proteins
– Some bacteria use SO42 or H2S
• Phosphorus
– In DNA, RNA, ATP, and membranes
– PO43 is a source of phosphorus
• Trace Elements
– Inorganic elements required in small amounts
– Usually as enzyme cofactors
• Oxygen (O2)
Obligate
aerobes
Faultative Obligate Aerotolerant
Microaerophiles
anaerobes anaerobes anaerobes
Toxic Forms of Oxygen
• Singlet oxygen: O2 boosted to a higher-energy state
• Superoxide free radicals: O2
• Peroxide anion: O22
• Hydroxyl radical (OH)
Biofilms
• Microbial
communities
• Form slime or
hydrogels
– Bacteria attracted by
chemicals via
quorum sensing
Figure 6.5
The Requirements for Growth:
Chemical Requirements
• Organic Growth Factors
– Organic compounds obtained from the
environment
– Vitamins, amino acids, purines, pyrimidines
Anaerobic Culture Methods
• Reducing media
– Contain chemicals (thioglycollate or oxyrase) that
combine O2
– Heated to drive off O2
An Anaerobic Chamber
Figure 6.7
Anaerobic Culture Methods
• Anaerobic jar
Figure 6.5
Biosafety Levels
• 1: No special precautions
• 2: Lab coat, gloves, eye protection
• 3: Biosafety cabinets to prevent airborne
transmission
• 4: Sealed, negative pressure
– Exhaust air is filtered twice
Biosafety Level 4 (BSL-4)
Laboratory
Figure 6.8
Selective Media
• Suppress unwanted
microbes and
encourage desired
microbes.
Figure 6.9b, c
Differential Media
• Make it easy to distinguish colonies of different
microbes.
Figure 6.9a
Enrichment Media
• Encourages growth of desired microbe
• Assume a soil sample contains a few phenoldegrading 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
• 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)
Streak Plate
Figure 6.10a, b
Preserving Bacteria Cultures
• Deep-freezing: -50°to -95°C
• Lyophilization (freeze-drying): Frozen (-54°
to -72°C) and dehydrated in a vacuum
Reproduction in Prokaryotes
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Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
Binary Fission
Figure 6.11
Figure 6.12b
If 100 cells growing for 5 hours produced
1,720,320 cells:
Figure 6.13
Phases of Growth
ANIMATION Bacterial Growth
Curve
Figure 6.15
Direct Measurements of Microbial
Growth
• Plate Counts: Perform serial dilutions of a
sample
Figure 6.15, top portion
• Inoculate Petri
plates from
serial dilutions
Plate Count
Figure 6.16
Plate Count
• After incubation, count colonies on plates that
have 25-250 colonies (CFUs)
Figure 6.15
•
Direct Measurements of Microbial
Growth
Filtration
Figure 6.17a, b
•
Direct Measurements of Microbial
Growth
Multiple
tube MPN
test
• Count
positive
tubes and
compare to
statistical
MPN table.
Figure 6.18b
Direct Measurements of Microbial
Growth
• Direct Microscopic Count
Direct Measurements of Microbial
Growth
Figure 6.19
•
Estimating Bacterial Numbers by
Turbidity Indirect Methods
Figure 620
Estimating Bacterial Numbers by
Indirect methods
• Metabolic activity
• Dry weight
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