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Ch. 6.

Microbial Nutrition & Growth
Learning Objectives:
• Chemical and energy requirements
– (autotrophs, heterotrophs, chemotrophs, phototrophs)
• Factors affecting growth
– (oxygen, temperature, pH, water availability)
• Culturing microbes and different growth media
– (selective, differential, anaerobic, etc.)
• Assessing microbial growth
– (calculating growth, growth curve, methods to measure growth)

• Two groups of organisms based on source of carbon
–Autotrophs: use inorganic source of carbon ( CO
2
)
–Heterotrophs: catabolize reduced organic
molecules as source of carbon


• Two groups of organisms based on use of chemicals
or light as source of energy
–Chemotrophs: acquire energy from redox reactions
using inorganic and organic compounds
–Phototrophs: use light as their energy source

Major factors influencing growth

– Oxygen
– Temperature
– pH
– Water availability
– Oxygen requirements

•Obligate aerobes: Bacteria must grow with
oxygen (essential for growth)

•Obligate anaerobes: Bacteria must grow
without oxygen (forms of oxygen are toxic to their
cell structure)

Environmental factors that influence microbes: e.g. gas needs
Oxygen can transform into toxic products:
Singlet oxygen (
1
O
2
), superoxide ion (O
2
-
), peroxides (H
2
O
2
), and
hydroxyls (OH
-
) can destroy cells
Most cells have enzymes to capture and neutralize these toxic
products
O
2
-
+ O
2
-
+ 2H
+
Superoxide dismutase H
2
O
2
+ O
2
H
2
O
2
+ H
2
O
2
Catalase 2H
2
O + O
2
Aerobic, facultative anaerobic organisms have these enzymes,
but anaerobic organisms do not have these enzymes
Catalase test: positive reaction is the presence of bubbles
Oxygen requirements
• Aerobes – undergo aerobic respiration

• Microaerophiles – aerobes that require oxygen
levels from 2 to 10%

• Facultative anaerobes – can perform
fermentation or anaerobic respiration or aerobic
respiration

• Aerotolerant anaerobes – do not use
aerobic metabolism but have some enzymes that
detoxify oxygen’s poisonous forms

• Anaerobes – do not use aerobic metabolism

Temperature

Each microbe has its own
optimum temperature for
growth

• Too high a
temperature
denatures proteins
and cell membranes
become too fluid

• Too low a temperature
results in rigid and
fragile membranes
Temperature

• Psychrophiles
-5°C to 15°C
• Arctic and Antarctic regions
• Psychrotrophs
20°C to 30°C
• Mesophiles
25°C to 45°C
• Thermophiles
45°C to 70°C
• Hot springs
• Hyperthermophiles
70°C to 110°C
• Usually members of Archaea
• Hydrothermal vents

Thermophilic bacteria
An example of a psychrophilic alga
pH

• Neutrophiles:
• bacteria and protozoa that grow best in a narrow
range around neutral pH (6.5-7.5)

• Acidophiles:
• bacteria and fungi that grow best in acidic
habitats,

• Alkalinophiles:
• live in alkaline soils and water up to pH 11.5
All microorganisms require water for growth

–Facultative halophiles: can tolerate high
salt environments

–Obligate halophiles: bacteria that must have
high salt for cell growth (up to 30% salt)

• E.g. Halobacterium (a member of Domain Archaea)
Water availability
– Physical effects of water

–Water exerts pressure in proportion to its depth

–Barophiles:
–organisms that live under extreme pressure

• Culturing (growing) microbes…..

• ‘Inoculum ‘ refers to the cells that you introduce into
medium (broth or solid)

• Pure culture: population of cells derived from single cell
– All cells genetically identical

• Pure culture obtained using aseptic technique

• Cells grown on culture media
– Can be broth (liquid) or solid form (agar plate/slant)
Isolation of cells in a mixed culture of bacteria:

Colony: formed from a single cell that undergoes
many cell divisions

Agar plate: agar is the solid medium required for
growth of the colony; agar comes from red algae
Obtaining Pure Culture
• Streak-plate method obtaining isolated colonies

– Object is to reduce number of cells being spread

• Each successive spread decreases number of cells per
streak
Pour plate method of obtaining isolated colonies
• Culture Media

– Types of media
• Defined media
• Complex media
• Selective media
• Differential media
• Anaerobic media


Defined (synthetic) media: exact chemical composition is
known and each batch is chemically identical
Blood agar is also a differential complex medium
Selective medium: selecting growth of some microbes and
inhibiting growth of other microbes.
Beta-hemolysis
Alpha-hemolysis
No hemolysis
(gamma-hemolysis)
Blood agar – used as a differential medium
Streptococcus pyogenes
Streptococcus pneumoniae
Enterococcus faecalis
MacConkey agar as a selective and differential medium
An anaerobic culture system
Clamp
Airtight lid
Envelope
containing
chemicals to
release CO
2

and H
2

Palladium pellets
to catalyze reaction
removing O
2

Methylene blue
(anaerobic
indicator)
Bacterial cells divide by binary fission
Doubling time/Generation time: time required for parent cell to
divide and produce two daughter cells


Principles of Bacterial Growth
Lag phase: growth lags; cells adjusting, not multiplying at max. rate
Log phase: exponential growth, max. rate of cell division
Stationary phase: cells stop growing/grow slowly; metabolic rate
declines; see depletion of nutrients, buildup of wastes
Death phase: number of viable cells decreases, rate depends on
species
The growth curve in a bacterial culture
• Growth can be calculated
• Example
– we have a culture of 3 cells in original
population
»assume 20 minute generation time
»after 2 hours of incubation the population
contains:
»3 x 2
n
where n is the number of generations
»3 x 2
6
= 3 x 64 = 192 cells


Principles of Bacterial Growth
Formula: initial # of cells x 2
n
=

# cells after growth
• Measuring Microbial Growth

• Viable plate counts
• Direct cell counts
• Membrane filtration
• Turbidity method


Estimating microbial population size- viable plate count method
Detecting Bacterial Growth
Viable plate counts

– Measures viable cells growing on solid culture media

– Count based on assumption that one cell gives rise to
one colony
• Number of colonies = number of cells in sample

– Ideal number to count
• Between 30 and 300 colonies

Direct cell count

– does not distinguish
between living and
dead cells

– bacterial cell number
is measured in a
known volume in a
hemocytometer

Membrane filtration used to estimate microbial population
- measures with
spectrophotometer

• measures light
transmitted through
sample
– Measurement is
inversely
proportional to cell
concentration

– Limitation
• Must have high number
of cells
Turbidity Method