Ch 6
Microbial
Growth
Objectives:
Classify microbes into five groups on the basis of preferred temperature
range.
Explain the importance of osmotic pressure to microbial growth.
Provide a use for each of the four elements (C, N, S, P) needed in large
amounts for microbial growth.
Explain how microbes are classified on the basis of O2 needs.
Identify ways in which aerobes avoid damage by toxic forms of O2.
Describe the formation of biofilms and their potential for causing infection.
Distinguish between chemically defined and complex media.
Justify the use of each of the following: anaerobic techniques, living
host cells, candle jars, selective, differential, and enrichment media.
Define colony and CFUs and describe how pure cultures can be isolated with
streak plates.
Explain how microbes are preserved by deep-freezing and lyophilization.
Distinguish between binary fission and budding.
Define generation time and explain the bacterial growth curve.
Review some direct and indirect methods of measuring bacterial cell growth.
Microbial Growth
Microbial growth: Increase in cell number, not cell size!
Physical Requirements for Growth:
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
Temperature
Five groups based on optimum growth temperature
1. Psychrophiles
2. Psychrotrophs
3. Mesophiles
4. Thermophiles
5. Hyperthermophiles
Fig. 6 .3
Fig 6.3: Effect of amount of food on its cooling rate
Physical Requirements for Growth:
pH and Osmotic Pressure
Most bacteria grow best between pH 6.5 and 7.5:
Neutrophils
Some bacteria are very tolerant of acidity or thrive
in it: Acidophiles (preferred pH range 1 to 5)
Molds and yeasts grow best between pH 5 and 6
Hypertonic environments (increased salt or sugar)
cause plasmolysis
Obligate halophiles vs.
facultative halophiles
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Fig 6.4
Chemical Requirements for Growth: Carbon,
N, S, P, etc.
Carbon
Half of dry weight
Chemoheterotrophs use organic carbon sources
Nitrogen, Sulfur, Phosphorus
Needed for ?
Found in amino acids and proteins
(most bacteria decompose proteins)
S in thiamine and biotin
Phosphate ions (PO43)
Vit B1
Vit B7
Also needed K, Mg, Ca, trace elements (as cofactors),
and organic growth factors
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Chemical Requirements for Growth: Oxygen
O2 requirements vary greatly
Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria
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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
Fig 6.5
Microbial communities form
slime or hydrogels
Starts via attachment of
planctonic bacterium to surface structure.
Bacteria communicate by chemicals via quorum sensing
Sheltered from harmful factors (disinfectants etc.)
Cause of most nosocomial infections
Clinical Focus: Delayed Bloodstream Infection Following
Catheterization
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Culture Media
Culture medium: Nutrients prepared for microbial growth
Have to be sterile (not contain living microbes)
Inoculum: Microbes introduced into medium
Culture: Microbes growing in/on culture medium
Chemically defined media: Exact chemical composition is known (for research purposes only)
Complex media: Extracts and digests of yeasts,
meat, or plants, e.g.:
Nutrient broth
Nutrient agar
Blood agar
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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
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Anaerobic Culture Methods
Use reducing media, containing chemicals (e.g.:
thioglycollate) that combine with O2
Are heated shortly before use to drive off O2
Use anaerobic jar
Novel method in clinical labs:
Add oxyrase to growth media
OxyPlate (no need for anaerobic jar)
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Fig 6.5
Capnophiles: Aerobic Bacteria Requiring High
CO2
Low oxygen, high CO2
conditions resemble those
found in
intestinal tract
respiratory tract and
other body tissues where
pathogens grow
E.g: Campylobacter jejuni
Use candle jar, CO2generator packets, or CO2
incubators
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Candle jar
Fig 6.7
Selective Media and Differential Media
Selective medium:
Additives suppress
unwanted and encourage
desired microbes e.g.
EMB, mannitol salt agar etc.
Differential medium:
changed in recognizable
manner by some bacteria
Make it easy to distinguish
colonies of different
microbes e.g. and
hemolysis on blood agar;
MacConkey agar, EMB,
mannitol salt agar etc.
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Enrichment Media/Culture
Encourages growth of desired microbe
Example: Assume 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
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Pure Cultures
Contain only one species or strain.
Most patient specimens and
environmental samples contain
several different kinds of bacteria
Streak-plate method is commonly used
Colony formation: A population of cells arising from
a single cell or spore or from a group of attached
cells (also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
Aseptic technique critical!
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Streak Plate Method
3 or 4
quadrant
methods
Preserving Bacterial Cultures
Deep-freezing: Rapid cooling of pure culture in
suspension liquid to 50to 95C. Good for several
years.
Lyophilization (freeze-drying): Frozen (54 to 72C)
and dehydrated in a vacuum. Good for many years.
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The Growth of Bacterial Cultures
Binary fission exponential growth
Budding
Generation time time required for cell to divide
(also known as doubling time)
Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)
Consider reproductive potential of E. coli
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Fig 6.13
Figure 6.12b
Bacterial Growth Curve
Illustrates the dynamics of growth
Foundation
Fig 6.15
Phases of growth
Lag phase
Exponential or
logarithmic (log) phase
Stationary phase
Death phase (decline phase)
Compare growth in liquid and on solid media
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Bacterial Growth Curve: Arithmetic vs.
Exponential Plotting
Fig 6.14
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Direct Measurements of Microbial Growth
Viable cell counts: Plate counts: Serial
dilutions put on plates CFUs form colonies
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Fig
6.16
Fig 6.17
Figure 6.15, step 1
Additional Direct Measurements
1. Filtration method of choice for low counts
2. Direct microscopic count: Counting chambers
(slides) for microscope
Fig 6.20
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Estimating Bacterial Numbers by Indirect
Methods
Spectrophotometry to measure turbidity
OD is function of cell number
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Measuring Microbial Growth - Overview
Direct Methods
Indirect Methods
Plate counts
Turbidity
Filtration
Metabolic activity
MPN
Dry weight
Direct microscopic count
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