Microbial Growth

1
Chapter 6

• Microbial Growth

2

• Microbial growth increase in number of cells,
  not cell size

3
What are

• Populations
• Colonies

4
The Requirements for Growth

• Physical requirements
• Temperature
• pH
• Osmotic pressure
• Chemical requirements
• Carbon
• Nitrogen, sulfur, and phosphorous
• Trace elements
• Oxygen
• Organic growth factor

5
The Requirements for Growth Physical Requirements

• Temperature
• Minimum growth temperature
• Optimum growth temperature
• Maximum growth temperature

6
Temperature
Figure 6.1
7
Psychrotrophs

• Grow between 0C and 20-30C
• Cause food spoilage

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Psychrotrophs
Figure 6.2
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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

10
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

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The Requirements for Growth Physical Requirements
Figure 6.4
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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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• Nitrogen
• 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

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• Trace Elements
• Inorganic elements required in small amounts
• Usually as enzyme cofactors

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• Oxygen (O2)

Obligate aerobes Faultative anaerobes Obligate anaerobes Aerotolerant anaerobes Microaerophiles

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Toxic Forms of Oxygen

• Singlet oxygen O2 boosted to a higher-energy
  state
• Superoxide free radicals O2?
• Peroxide anion O22?
• Hydroxyl radical (OH?)

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Biofilms

• Microbial communities
• Form slime or hydrogels
• Bacteria attracted by chemicals via quorum
  sensing

Figure 6.5
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The Requirements for Growth Chemical Requirements

• Organic Growth Factors
• Organic compounds obtained from the environment
• Vitamins, amino acids, purines, pyrimidines

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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 100C
• Solidifies 40C

21
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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Culture Media
Table 6.2 6.4
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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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An Anaerobic Chamber
Figure 6.7
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Anaerobic Culture Methods

• Anaerobic jar

Figure 6.5
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Capnophiles require high CO2

• Candle jar
• CO2-packet

Figure 6.7
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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

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Biosafety Level 4 (BSL-4) Laboratory
Figure 6.8
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Selective Media

• Suppress unwanted microbes and encourage desired
  microbes.

Figure 6.9b, c
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Differential Media

• Make it easy to distinguish colonies of different
  microbes.

Figure 6.9a
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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
• 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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• 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
Figure 6.10a, b
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Preserving Bacteria Cultures

• Deep-freezing -50to -95C
• Lyophilization (freeze-drying) Frozen (-54 to
  -72C) 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
Figure 6.11
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Figure 6.12b
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• If 100 cells growing for 5 hours produced
  1,720,320 cells

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Figure 6.13
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Phases of Growth
ANIMATION Bacterial Growth Curve
Figure 6.15
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Direct Measurements of Microbial Growth

• Plate Counts Perform serial dilutions of a sample

Figure 6.15, top portion
42
Plate Count

• Inoculate Petri plates from serial dilutions

Figure 6.16
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Plate Count

• After incubation, count colonies on plates that
  have 25-250 colonies (CFUs)

Figure 6.15
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Direct Measurements of Microbial Growth

• Filtration

Figure 6.17a, b
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Direct Measurements of Microbial Growth

• Multiple tube MPN test
• Count positive tubes and compare to statistical
  MPN table.

Figure 6.18b
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Direct Measurements of Microbial Growth

• Direct Microscopic Count

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Direct Measurements of Microbial Growth
Figure 6.19
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Estimating Bacterial Numbers by Indirect Methods

• Turbidity

Figure 620
49
Estimating Bacterial Numbers by Indirect methods

• Metabolic activity
• Dry weight