Bacterial Growth Flashcards
Growth Requirements
➢ Elements that are required for bacterial growth
➢ Growth factors that are required for bacterial growth.
➢ Carbon sources and energy sources for bacterial growth.
➢ Biochemical and biophysical environment requirements
➢ Biochemical environment (culture media - classification and different types)
➢ Liquid versus solid
➢ Chemically defined/minimal media versus undefined/complex media
➢ Selective, differential, enrichment media
➢ Biophysical environment ( oxygen, pH, temperature, etc)
What are the growth factors?
➢ Purines and pyrimidines: Nucleic acid synthesis.
➢ Amino acids: Protein synthesis.
➢ Vitamins: Needed as coenzymes and functional groups of certain enzymes.
✓ Folic acid, biotin, nicotinic acid, vitamin B12, vitamin K etc.
Why do microorganisms need growth factors?
➢They are not able to synthesize themselves because they don’t have
the metabolic pathway(s) for their production.
➢ Growth factors are essential for bacterial metabolism, growth, and
reproduction
➢ They are required in small amounts by cells (because they fulfill specific
roles in biosynthesis).
Carbon and Energy Sources for Bacterial Growth
For growth in nature or in the laboratory, a bacterium must have:
➢An energy source
➢A carbon source
➢Any other required nutrients
➢A permissive range of physical conditions:
✓ O2 concentration
✓ Temperature
✓ pH
Producer
they can make their
own food from raw
materials and energy
Consumer
they consume
producers or other
consumers.
The biochemical (nutritional) environment
The nutrients that are made available as a culture medium
Culture media for the growth of bacteria
Liquid medium:
Used for growth of pure batch
cultures.
Solid medium:
Isolation of pure cultures, estimation of viable bacterial populations etc.
➢ Agar, a complex polysaccharide, is mainly used.
➢ Gelling properties and inability of most microbes to metabolise it make
it an ideal support medium
Classification of growth media
1- Chemically-defined (synthetic) media:
➢ Media of which the exact chemical composition
is known, including any growth factors.
➢ Also known as a minimal medium
2- Complex (undefined) media:
➢ Media of which the exact chemical constitution
of the medium is not known.
➢ Usually contain complex materials of biological
origin such as blood or milk or yeast extract or
beef extract
Other Medium Types
Example: Mannitol Salts Agar (both selective and differential)
(Selective = the medium will only allow certain microorganisms to grow.)
(Differential = the medium will show some characteristic of the microorganisms growing on it and can be used to
differentiate between different species.)
.Mannitol salts medium
contains 7.5% NaCl (salt),
whereas typical media
contains about 0.5% NaCl.
. Only halophilic (salt-loving
species) or halotolerant (salt-
tolerant) species to grow on it.
.Mannitol salts medium contains
mannitol (a sugar). If the
bacteria ferments mannitol acid
is produced.
.If a species ferments mannitol and produces acid, the pH of the medium decreases and phenol red will become yellow.
.The medium contains an acid
indicator (phenol red). This
indicator is red when the pH is
neutral.
3- Enrichment media (liquid version of the selective media):
Contains some component that encourages the growth of one microbe over others.
The biophysical environment means oxygen, pH, temperature, etc
Bacteria and Archaea grow under a staggering range of conditions.
Important conditions include:
* Oxygen
* pH
* Temperature
* Salinity
* Osmolarity and water activity
Oxygen
Bacteria and Archaea display a wide range of responses to molecular oxygen (O2.)
Obligate aerobes: Require O2 for growth.
Obligate anaerobes: Do not use O2. O2 is toxic to these organisms.
Facultative anaerobes: Can switch between aerobic and anaerobic types of
metabolism.
Aerotolerant anaerobes: Use an exclusively anaerobic (fermentative) type of
metabolism but they are insensitive to the presence of O2.
Microaerophile: Oxygen required but only at low levels.
OXYGEN and METABOLISM
Aerobic respiration: Oxygen is the terminal electron acceptor. Molecular
oxygen is a highly oxidizing agent and, therefore, is an excellent electron acceptor.
Anaerobic respiration: Less-oxidizing substances such as sulfate (SO42−),
nitrate (NO3−), sulfur (S), or fumarate are used. They have smaller reduction potentials than O2, meaning that less energy is released per oxidized molecule.
Fermentation: Releases energy from a sugar or other organic molecule,
does not require oxygen or an electron transport system, and uses an
organic molecule as the final electron acceptor
DETERMINING OXYGEN REQUIREMENTS
Thioglycolate broth (a
differential medium).
(Differential = the medium will
show some characteristic of the
microorganisms growing on it and
can be used to differentiate
between different species.)
Sodium thioglycolate in the
medium consumes oxygen,
since oxygen diffuses from
the top of the broth and as
such a range of oxygen
concentrations is generated.
pH
pH is a measure of the hydrogen ion concentration which varies from <0.5 in acidic soils to >10.5 in alkaline lakes.
Acidophiles: Grow in acid environments.
Neutrophiles: Grow around neutral pH.
Alkaliphiles: Grow under alkaline conditions.
Temperature
Microorganisms have been found growing in virtually all environments
where there is liquid water, regardless of its temperature.
Psychrophiles: Cold-loving -20°C to +10°C
Mesophiles: Around 20oC-45°C
Thermophiles: 45oC-70°C
Extreme thermophiles: 80oC - >120°C
Water Activity
1- Water is the solvent in which the molecules of life are dissolved,
2- The availability of water is therefore a critical factor that affects the growth of all cells.
3- The availability of water for a cell depends upon its presence in the atmosphere (relative humidity) or its
presence in solution or a substance (water activity).
4- The water activity (Aw) of pure H2O is 1.0 (100% water).
5- Water activity is affected by the presence of solutes such as salts or sugars, that are dissolved in the
water.
6- The higher the solute concentration of a substance, the lower is the water activity and vice-versa.
7- Microorganisms live over a range of Aw from 1.0 to 0.7. The Aw of human blood is 0.99; seawater = 0.98;
maple syrup = 0.90; Great Salt Lake = 0.75. Water activities in agricultural soils range between 0.9 and 1.0
The Bacterial Growth Cycle
1- Going through these phases depends on
availability of growth requirements (biochemical and
biophysical).
2- If a bacterium in stationary phase is put in fresh
media full of nutrients, it can go into exponential phase again
Lag Phase
The Lag phase:
➢ Cells are metabolically
active and increase in
size.
➢ Protein and enzyme
synthesis occurs but
there is no cell division
The Acceleration phase (Comes under lag phase)
The cells begin to divide
Factors that affect the Lag Phase
1- State of the cells
Actively growing cells will start to divide
faster than stationary phase cells.
2- Type of medium
Cells grown in a rich medium then put in a
minimal medium will lag because they have to
synthesise many enzymes for e.g. amino-acid
synthesis.
3- Physiological conditions
4- The nature of the organism
The Exponential (log) phase
The Exponential phase:
All the cells are dividing at their
maximum rate but
asynchronously
Factors that affect the Exponential Phase
1- Availability of nutrients
The length of the exponential phase
depends on the availability of nutrients. Rich
media will usually support more growth than
minimal media. The final cell density may reach >1010 cells ml-1.
2- Buffering capacity of the medium
Metabolic by products lead to changes in pH
and these changes can lead to inhibition of growth.
3- Build-up of toxins or depletion of oxygen
Prevention of these can extend the growth phase
The maximum rate of growth in any medium is known as the specific
growth rate, μ (EXPONENTIAL PHASE)
number of divisions per h
time taken to double (EXPONENTIAL PHASE)
Mean generation time or Mean
doubling time.
➢ This can vary from as little as 8-10 min to as long as 24 h+.
➢ E. coli will divide every 20 min in a rich medium but may take as long as
60 min+ in a poor or minimal medium.
➢ Growth rate is affected by physiological conditions
Exponential Growth
➢ The increase in cell number in an exponentially growing cell culture is a geometric
progression of the number 2.
➢ Where N is the final cell number, N0 is the initial cell number and n is the number
of generations.
➢ If we know the initial and final cell numbers, we can calculate n.
N = N02n
Example (N = N02n) If we know the initial and final cell numbers, we can calculate n
1) 128,000,000 = 1,000,000 * 2n
2)128,000,000/1,000,000 = 2n
3)128= 2n
4)og2 (128) = n
n = 7
Explanation: In only 7
divisions 1x10^6 cells become
1.28X10^8
Generation Time
➢ The time interval for one cell division is called the generation time.
➢ Where g is the generation time, t is the time taken for that number of generations
and n is the number of generations.
g = t/n
Example of g = t/n. If we know n (how many times the bacteria doubled) and t (in how many minutes) we
can calculate g (generating time)
7 divisions for this bacteria took 140 minutes. What is the generation time for this bacteria?
1)g = t/n
2)g = 140/7
3)g = 20
How it can be explained:
The generation time of this bacteria is 20 minutes.
The bacteria number doubles/divides every 20 minutes
Stationary Phase
1) The Deceleration phase:
Cells begin to stop dividing
because of lack of nutrients, build-up of toxic metabolites,
lack of oxygen or lack of space (the latter is rare in the case of bacteria)
2) The Stationary phase:
The cells either cease dividing
or there is a slow rate of cell
division matched by slow cell
death. Metabolism still occurs.
Secondary metabolites such as
antibiotics are produced in this
phase
Factors that affect the Stationary Phase
1- Temperature
Some bacteria will exist in the stationary phase
for prolonged periods, especially if refrigerated e.g.
E. coli will last for 6 months to a year on an agar
slope, whereas Myxococcus xanthus will die off
rapidly unless preserved e.g. by freezing in liquid
nitrogen.
2- The nature of the organism
Some bacteria begin to autolyse as soon as they
enter stationary phase – and therefore have a very
short stationary phase.
3- The presence of toxins
Presence of acid metabolites or NH3 shortens
the stationary phase and speeds cell death.
Death/Decline Phase
Autolysis occurs and the cells
begin to lyse.
Eventually nutrients released may
allow surviving cells to enter
another growth cycle.
Factors that affect the Death Phase
Nature of the organism
Cells that enter the death phase may avoid lysis
by either entering a “Viable but non-culturable
(VNC)” state.
These VNC state cells are metabolically active
but will not grow and divide on ordinary culture
media.
Alternatively, the cells may produce resting,
metabolically inactive forms (spores or cysts).
Bacterial Growth Methods
1) Batch culture: Such as in a tube or flask. exponential phase is limited. In
a batch culture the culture medium is continually being altered by the
metabolic activity of the growing cells
2) The chemostat is a continuous culture device. It stops cultures from reaching stationary phase and
keeps them in exponential phase. Controls both the growth rate and population density of the
culture. Two factors are important for this control: the dilution rate and the concentration of a
limiting nutrient.
Measuring bacterial growth
1 - Direct measurement:
Total count: Direct microscope count. Reasonably quick. Cannot
distinguish dead from live cells. Not good for low density cultures.
Viable count: Dilution of culture followed by plating on suitable
agar medium. Probably best method but laborious.
2- Indirect measurement:
Turbidity measurement: Effectively measure the “cloudiness” or
turbidity of a culture. Uses a spectrophotometer to measure the
optical density (OD) of the culture.
Bacterial Cell Growth (BINARY FISSION)
➢ The cell enlarges to twice the size.
➢ DNA is replicated.
➢ FtsZ (Filament temperature sensitive) protein locates as a ring in
the centre of the cell.
➢ Cell septum is formed at the site of the FtsZ ring.
➢ Cell divides.
DNA replication and cell division
DNA replication must be coordinated with cell division.
Thus, to divide every 20 minutes E. coli must start replicating its DNA
every 20 minutes.
However, it would take about an hour for E. coli to replicate its
genome.
How can this be done?
Multifork Replication
oriC is the origin of replication, terC is the termination point
