BACTERIAL GROWTH
Industrial Microbiology
BACTERIAL GROWTH
4-2
TERMS.
 Binary fission
 Generation time
 Phases of growth
BINARY FISSION
Figure 4.2
1.
Prokaryote cells grow
by increasing in cell
number (as opposed to
increasing in size).
2.
Replication is by binary
fission, the splitting of
one cell into two
3.
Therefore, bacterial
populations increase by
a factor of two (double)
every generation time.
GENERATION TIME
(doubling time) in number.
 Ex. Escherichia coli (E. coli) double every 20
minutes
 Ex. Mycobacterium tuberculosis double every 12
to 24 hours
4-4
 The time required to for a population to double
PRINCIPLES OF BACTERIAL GROWTH
 Growth can be calculated
 Nt = N0 x 2n
(Nt ) number of cells in population
 (N0 ) original number of cells in the population
 (n) number of divisions
 Example

N0 = 10 cells in original population
 n = 12
 4 hours assuming 20 minute generation time
 Nt = 10 x 212
 Nt = 10 x 4,096
 Nt = 40,960

Growth in Batch Culture
1. Bacteria growing in batch culture produce a growth
curve with up to four distinct phases.
2. Batch cultures are grown in tubes or flasks and are
closed systems where no fresh nutrients are added or
waste products removed.
3. Lag phase occurs when bacteria are adjusting to the
medium. For example, with a nutritionally poor
medium, several anabolic pathways need to be turned
on, resulting in a lag before active growth begins.
GROWTH IN CULTURE
4. In log or exponential phase, the cells are growing as
fast as they can, limited only by growth conditions and
genetic potential. During this phase, almost all cells are
alive, they are most nearly identical, and they are most
affected by outside influences like disinfectants.
5. Due to nutrient depletion and/or accumulation of toxic
end products, replication stops and cells enter a
stationary phase where there is no net change in cell
number.
6. Death phase occurs when cells can no longer maintain
viability and numbers decrease as a proportion.
LAB CHEMOSTAT SYSTEM
ENVIRONMENTAL FACTORS
Oxygen requirement
pH
Water availability
4-10
Temperature
TEMPERATURE
4-11
 Enzymes, the machinery of the cell, are influenced by
external factors and can be shown to have a range where
they function that includes an optimal value that produces
the highest activity.
 The range of enzyme activity determines the range for
growth of specific bacteria, analogously leading to a value
for optimal growth rate.
 In the case of temperature, bacteria are divided into
categories based on the temperature range where they can
grow and the temperature that provides optimal growth.
TEMPERATURE
 Psychrophile

0o to 18o C
 Psychrotroph

20°C to 30°C

Important in food spoilage
 Mesophile

25°C to 45°C
More common
 Disease causing

 Thermophiles

45°C to 70°C

Common in hot springs and hot water heaters
 Hyperthermophiles

70°C to 110°C
Live at very high temperatures, high enough where water threatens to
become a gas
 Usually members of Archaea
 Found in hydrothermal vents

OXYGEN REQUIREMENTS
4-13
•
Oxygen is a very reactive molecule and can affect cells in
several ways. The effect of oxygen is often determined using
thioglycollate broth, a special medium that contains a
reducing agent (thioglycollate) that removes oxygen so that
a gradient occurs within the tube.
OXYGEN REQUIREMENTS
•
Microaerophiles require oxygen for growth but the 20% in air
is too toxic. As a result, they grow near the top but beneath
the surface of thioglycollate broth where the oxygen
concentration is typically 4 – 10%.
•
Facultative anaerobes can use oxygen for aerobic respiration
but can switch to fermentative metabolism in the absence of
oxygen. As a result, they will grow throughout thioglycollate
broth. (Heavier growth at top.)
•
Aerotolerant anaerobes are anaerobic bacteria that can grow
in the presence of air
OXYGEN REQUIREMENTS
•
Obligately aerobic bacteria can obtain energy only
through aerobic respiration and have to have
oxygen available. Thus, they will grow only at the
surface of thioglycollate broth.
•
Obligately anaerobic bacteria die in the presence
of oxygen and can only grow at the bottom of
thioglycollate broth.
PH




Neutrophiles grow best around neutral pH (7)
Acidophiles grow best at pH < 7
Alkophiles grow best at pH > 7
Acidotolerant grow best at pH 7 but can also
grow at lower pH
Alkotolerant grow best at pH 7 but can also
grow at higher pH
4-16

WATER ACTIVITY
 Liquid water is essential for life.
 Aqueous solutions actually have different amounts of water
available, depending on how many solutes are dissolved in it.
As a very simple model, consider two glasses, one full of pure
water, the other containing the same amount of water plus a
sponge. Which one would be easier to drink? On a much
smaller scale, dissolved solutes act like a sponge, making less
water available.
 Water activity (aw) can be decreased by the addition of any
soluble molecule although salt (NaCl) and sugars are probably
the most common.
WATER ACTIVITY

Microbes that require a high water activity are termed
nonhalophiles. (Halophile = salt-loving)

Some bacteria require salt to grow and are called halophiles.
If a very high concentration of salt is required (around
saturation), the organisms are termed extreme halophiles.

A nonhalophile that can grows best with almost no salt but can
still grow with low levels of salt (~ 7%) is called halotolerant.

In general, fungi are more tolerant of low water
activity. (That’s why your jelly is more likely to get
contaminated by fungi than bacteria.)
NUTRITIONAL REQUIREMENTS
 Growth of prokaryotes depends on nutritional
factors as well as physical environment
 Main factors to be considered are:
Required elements
 Growth factors
 Energy sources
 Nutritional diversity

NUTRITIONAL REQUIREMENTS
 Major elements (CHONPS + K, Mg, Fe, Ca)

Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium,
magnesium, iron, and calcium

Essential components for macromolecules
 Organisms classified based on carbon usage

Heterotrophs


Use organic carbon as nutrient source
Autotrophs

Use inorganic carbon (CO2) as carbon source
 Trace elements (Co, Cu, Ni, Zn, Se, Mg, Wo)

Cobalt, zinc, copper, molybdenum and manganese
Required in minute amounts
 Assist in enzyme function

 Nutritional diversity

Different organisms require the same nutrients but may require
different forms of the nutrients
MAJOR ELEMENTS
Element
% dry wgt
Source
Carbon
50
organic compounds or CO2
Oxygen
20
H2O, organic compounds, CO2, and O2
Nitrogen
14
NH3, NO3, organic compounds, N2
Hydrogen
8
H2O, organic compounds, H2
Phosphorus
3
inorganic phosphates (PO4)
Sulfur
1
SO4, H2S, So, organic sulfur compounds
Potassium
1
Potassium salts
Magnesium
0.5
Magnesium salts
Calcium
0.5
Calcium salts
Iron
0.2
Iron salts
CARBON SOURCE
 Organic molecules

Heterotrophs
 Inorganic carbon (CO2)

Autotrophs
NITROGEN SOURCE
 Organic nitrogen

Primarily from the catabolism of amino acids
 Oxidized forms of inorganic nitrogen

Nitrate (NO32-) and nitrite (NO2-)
 Reduced inorganic nitrogen

Ammonium (NH4+)
 Dissolved nitrogen gas (N2) (Nitrogen fixation)
PHOSPHATE SOURCE
 Organic phosphate
 Inorganic phosphate (H2PO4- and HPO42-)
SULFUR SOURCE
 Organic sulfur
 Oxidized inorganic sulfur

Sulfate (SO42-)
 Reduced inorganic sulfur

Sulfide (S2- or H2S)
 Elemental sulfur (So)
GROWTH FACTORS
 Some bacteria cannot synthesize some cell
constituents
 These must be added to growth environment

Referred to as growth factors
 Organisms can display wide variety of factor
requirements
 Some need very few while others require many

These termed fastidious
 Typical molecules
 Amino acids
 Nucleotide bases
 Enzymatic cofactors or “vitamins”
CULTURE MEDIA
 Chemically defined (all concentrations are
known)
 Selective (favors the growth of a particular
organism or group of organisms)
 Differential (has reactions that give isolates
different appearance)
 Anaerobic (oxygen-free)
4-29
 Complex (contains undefined components)
4-30
CHARACTERISTICS OF MEDIA
ENUMERATION OF BACTERIA
For unicellular microorganisms, such as bacteria, the
reproduction of the cell reproduces the entire organism.
Therefore, microbial growth is essentially synonymous
with microbial reproduction. To determine rates of
microbial growth and death, it is necessary to
enumerate microorganisms, that is, to determine their
numbers.
It is also often essential to determine the number of
microorganisms in a given sample. For example, the
ability to determine the safety of many foods and drugs
depends on knowing the levels of microorganisms in
those products.
CONTD
A variety of methods has been developed for the
enumeration of microbes.
These methods measure cell numbers, cell mass, or
cell constituents that are proportional to cell
number.
The four general approaches used for estimating
the sizes of microbial populations are direct and
indirect counts of cells and direct and indirect
measurements of microbial biomass.
METHODS
Direct Count of Cells : Cells are counted directly
under the microscope or by an electronic
particle counter
 Direct Count Using a Counting ChamberDirect
microscopic counts are performed by spreading a
measured volume of sample over a known area of
a slide, counting representative microscopic
fields, and relating the averages back to the
appropriate volume-area factors.
1.
CONTD
 Specially constructed counting chambers, such as
the Petroff-Hauser and Levy counting chambers,
simplify the direct counting procedure because
they are made with depressions in which a known
volume overlies an area that is ruled into squares.
 The ability to count a defined area convert the
numbers observed directly to volume makes the
direct enumeration procedure relatively easy.
CONTD’
2. Indirect Count of Cells : Microorganisms in a
sample are diluted or concentrated and grown on a
suitable medium; the development of growing
microorganisms (for example, colony formation on
agar plates) is then used to estimate the numbers of
microorganisms in the original sample
3. Direct Measurement of Microbial Biomass : Cell
mass is determined directly by weighing whole cells;
biomass can be correlated with cell numbers by
reference to a standard curve. Wet weight or dry
weight of bacteria may be used for estimation of cell
numbers of microorganisms in the original sample
CONTD’ `
4. Indirect Measurement of Microbial Biomass
Microbial biomass is estimated by measuring
relatively constant biochemical components of
microbial cells, such as protein, ATP,
lipopolysaccharides, peptidoglycan, and chlorophyll;
biomass can also be indirectly estimated by measured
turbidity that can then be correlated with cell
numbers by reference to a standard curve.` ``