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 oxygens 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
-5C to 15C
Arctic and Antarctic regions
Psychrotrophs
20C to 30C
Mesophiles
25C to 45C
Thermophiles
45C to 70C
Hot springs
Hyperthermophiles
70C to 110C
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