Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.

U
BACTERIA
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

BACTERIA- B.Sc SEM I

GROWTH MEASUREMENT CULTURE
Bacteria are prokaryotic organisms that most commonly replicate by the asexual
process of binary fission. These microbes reproduce rapidly at an exponential rate under
favorable conditions. When grown in culture, a predictable pattern of growth in a bacterial
population occurs. This pattern can be graphically represented as the number of living cells in
a population over time and is known as a bacterial growth curve. Bacterial growth cycles in
a growth curve consist of four phases: lag, exponential (log), stationary, and death.

 The bacterial growth curve represents the number of live cells in a bacterial
population over a period of time.
 There are four distinct phases of the growth curve: lag, exponential (log), stationary,
and death.
 The initial phase is the lag phase where bacteria are metabolically active but not
dividing.
 The exponential or log phase is a time of exponential growth.
 In the stationary phase, growth reaches a plateau as the number of dying cells equals
the number of dividing cells.
 The death phase is characterized by an exponential decrease in the number of living
cells

Bacteria require certain conditions for growth, and these conditions are not the same for all
bacteria. Factors such as oxygen, pH, temperature, and light influence microbial growth.
Additional factors include osmotic pressure, atmospheric pressure, and moisture availability.
A bacterial population's generation time, or time it takes for a population to double, varies
between species and depends on how well growth requirements are met.

Phases of the Bacterial Growth Cycle

In nature, bacteria do not experience perfect environmental conditions for growth. As such,
the species that populate an environment change over time. In a laboratory, however, optimal
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

conditions can be met by growing bacteria in a closed culture environment. It is under these
conditions that the curve pattern of bacterial growth can be observed.

The bacterial growth curve represents the number of live cells in a bacterial population over
a period of time.

 Lag Phase: This initial phase is characterized by cellular activity but not growth. A
small group of cells are placed in a nutrient rich medium that allows them to
synthesize proteins and other molecules necessary for replication. These cells increase
in size, but no cell division occurs in the phase.
 Exponential (Log) Phase: After the lag phase, bacterial cells enter the exponential or
log phase. This is the time when the cells are dividing by binary fission and doubling
in numbers after each generation time. Metabolic activity is high as DNA, RNA, cell
wall components, and other substances necessary for growth are generated for
division. It is in this growth phase that antibiotics and disinfectants are most effective
as these substances typically target bacteria cell walls or the protein synthesis
processes of DNA transcription and RNA translation.
 Stationary Phase: Eventually, the population growth experienced in the log phase
begins to decline as the available nutrients become depleted and waste products start
to accumulate. Bacterial cell growth reaches a plateau, or stationary phase, where the
number of dividing cells equal the number of dying cells. This results in no overall
population growth. Under the less favorable conditions, competition for nutrients
increases and the cells become less metabolically active. Spore forming bacteria
produce endospores in this phase and pathogenic bacteria begin to generate
substances (virulence factors) that help them survive harsh conditions and
consequently cause disease.
 Death Phase: As nutrients become less available and waste products increase, the
number of dying cells continues to rise. In the death phase, the number of living cells
decreases exponentially and population growth experiences a sharp decline. As dying
cells lyse or break open, they spill their contents into the environment making these
nutrients available to other bacteria. This helps spore producing bacteria to survive
long enough for spore production. Spores are able to survive the harsh conditions of
the death phase and become growing bacteria when placed in an environment that
supports life.

Nt = N0 x 2n (1)

Nt = cell number at time t, N0 = starting number at time zero,
n = number of doublings (generations).

Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial population is by

geometric progression. If we start with one cell, when it divides, there are 2 cells in the
first generation, 4 cells in the second generation, 8 cells in the third generation, and so on.
The generation time is the time interval required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)

Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

G = t/n,

t = time interval in hours or minutes, B = number of bacteria at the beginning of a time

interval, b = number of bacteria at the end of the time interval, n = number of generations
(number of times the cell population doubles during the time interval), b = B x 2n (This
equation is an expression of growth by binary fission)

Solve for n:
logb = logB + nlog2
n = logb - logB
           log2
n = logb - logB
           .301
n = 3.3 logb/B
G = t/n
Solve for G
G =        t
       3.3 log b/B

FACTORS THAT AFFECT BACTERIAL GROWTH

Water and Dampness

Warmth, moisture, pH levels and oxygen levels are the four big physical and chemical factors
affecting microbial growth. In most buildings, warmth and moisture are the biggest overall
issues present. Dampness is a big player in the growth of fungi. Just like any living thing,
water is essential to the life of microbes. They cannot multiply and spread without a
consistent water source available to them. Bathrooms and basements tend to be prone to
dampness and stagnant water, making them a key location for potential microbe issues. Leaks
in the ceiling from rainfall or from pipes in the water system that aren’t tended to can not
only damage equipment but can also be breeding grounds for microbes in areas that are
harder to notice or clean up.
Temperature
The temperature of an area can be a massive contributor to microbial growth. Bacteria thrives
in warmth, growing the most in areas close to the temperature of a human’s body. Cooler
locations tend to slow growth of microbes, as seen when food is refrigerated to keep it safe to
eat longer. Boiler rooms, rooms with heat-generating equipment and areas near heating vents
can nest bacteria and molds. Areas around machinery that create humidity are, of course,
locations that can cause the most concern when one is minding the health of a building.
Having an environmental consultant assess areas around equipment with heat as a byproduct
can help you prevent potential health issues before they even develop by creating solutions
through methods like improving airflow with optimized air ventilation. Temperature is
another important factor for bacterial growth. Bacteria that grow best in cooler environments
are called psycrophiles. These microbes prefer temperatures ranging between 4°C and 25°C
(39°F and 77°F). Extreme psycrophiles thrive in temperatures below 0°C/32°F and can be
found in places such as arctic lakes and deep ocean waters.
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

Bacteria that thrive in moderate temperatures (20-45°C/68-113°F) are called mesophiles.

These include bacteria that are part of the human microbiome which experience optimum
growth at or near body temperature (37°C/98.6°F).
Thermophiles grow best in hot temperatures (50-80°C/122-176°F) and can be found in hot
springs and geothermal soils. Bacteria that favor extremely hot temperatures
(80°C-110°C/122-230°F) are called hyperthermophiles.

Environmental pH:

The pH level of an environment can either help or hurt the growth of microbes. Microbes
tend to prefer pH levels that are neutral and are often harmed when more base or acidic
elements are present in a location. It’s for this reason that cleaning solutions, often highly
acidic, kill off bacteria effectively when used. This means that improving cleaning regimens
in a business’s building can greatly reduce the number of microbes growing and deter future
growths when performed regularly.
Another important factor for bacterial growth is pH. Acidic environments have pH values that
are less that 7, neutral environments have values at or near 7, and basic environments have
pH values greater than 7. Bacteria that are acidophiles thrive in areas where the pH is less
than 5, with an optimal growth value close to a pH of 3. These microbes can be found in
locations such as hot springs and in the human body in acidic areas such as the vagina.
The majority of bacteria are neutrophiles and grow best in sites with pH values close to
7. Helicobacter pylori is an example of a neutrophile that lives in the acidic environment of
the stomach. This bacterium survives by secreting an enzyme that neutralizes stomach acid in
the surrounding area. Alkaliphiles grow optimally at pH ranges between 8 and 10. These
microbes thrive in basic environments such as alkaline soils and lakes.

Oxygen and Nutrients:

Oxygen-enriched locations and areas with vital nutrients will cultivate more microbial growth
than locations with reduced oxygen levels. Controlling oxygen levels in an area can be
difficult, but keeping areas clear of food and other sources of nutrients will starve out bacteria
and keep a building clear of other pests as well. Bacteria can be categorized based on
their oxygen requirement or tolerance levels. Bacteria that can not survive without oxygen
are known as obligate aerobes. These microbes are dependent upon oxygen, as they convert
oxygen to energy during cellular respiration. Unlike bacteria that require oxygen, other
bacteria can not live in its presence. These microbes are called obligate anaerobes and their
metabolic processes for energy production are halted in the presence of oxygen.
Other bacteria are facultative anaerobes and can grow with or without oxygen. In the
absence of oxygen, they utilize either fermentation or anaerobic respiration for energy
production. Aerotolerant anerobes utilize anaerobic respiration but are not harmed in the
presence of oxygen. Microaerophilic bacteria require oxygen but only grow where oxygen
concentration levels are low. Campylobacter jejuni is an example of a microaerophilic
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

bacterium that lives in the digestive tract of animals and is a major cause of foodborne
illness in humans.
LIGHT
Some bacteria require light for growth. These microbes have light-capturing pigments that
are able to gather light energy at certain wavelengths and convert it to chemical
energy. Cyanobacteria are examples of photoautotrophs that require light for photosynthesis.
These microbes contain the pigment chlorophyll for light absorption and oxygen production
through photosynthesis. Cyanobacteria live in both land and aquatic environments and can
also exist as phytoplankton living in symbiotic relationships with fungi (lichen), protists, and
plants. 
Other bacteria, such as purple and green bacteria, do not produce oxygen and utilize sulfide
or sulfur for photosynthesis. These bacteria contain bacteriochlorophyll, a pigment capable
of absorbing shorter wavelengths of light than chlorophyll. Purple and green bacteria inhabit
deep aquatic zones.

Measurements of microbial growth:

There are various ways to measures microbial growth for the determination of growth rates
and generation times. For the measurement of growth either mass or population number is
followed because growth leads to increase in both. Growth can be measured by one of the
following types of measurements:

 Cell count this method involves the measurement of growth either by microscopy or

by using an electronic particle counter or indirectly by a colony count.
 Cell mass in this growth can be measured directly by weighing or by a measurement
of nitrogen concentration in cells or indirectly by the determination of turbidity
using spectrophotometer.
 Cell activity in this growth can be measured indirectly by analysis of the degree of
biochemical activity to the size of population.
The most obvious way to count microbial numbers is through:
Direct counting.
Petroff-hausser counting is one of the easiest and accurate way to count bacteria.
 Side view of the chamber showing the cover glass and the space beneath it that holds
a bacterial suspension.
 A top view of the chamber. The grid is located in the center of the slide.
 An enlarged view of the grid. The bacteria in several of the central squares are
counted, usually at X400 to X500 magnification.
 Concentration of the cells can be calculated by using the average no. of bacteria the
avg. number of bacteria in these squares.
 There are 25 squares covering a part of area of 1 mm2, then the entire number of
bacteria in 1 mm2 of the chamber is (number/square) (25 squares).
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

The chamber is 0.02 mm deep and thus, bacteria/mm3 = (bacteria/square) (25

squares) (50). The amount of bacteria per cm3 is 103 times this value. For example,
imagine the average count per square is 28 bacteria: bacteria/cm3 = (28 bacteria) (25
squares) (50) (103) = 3.5X 107.

Electronic enumeration of cell numbers

 In this method of microbial growth measurement, bacterial suspension is kept inside
an electronic particle counter, within which the bacteria are passed through tiny
orifice 10 to 30 μm in diameter.
 This orifice is then connected to the two compartments of the counter which contains
an electrically conductive solution.
 The electrical resistance between two compartments will increases momentarily,
when bacterium passes through the orifice. This generates an electrical signal which is
automatically counted.
 The main disadvantage of this method is that there is no way to determine whether the
cell counted is viable or not.
The plate count method

 This method allows the determination of the number of cells that will multiply under
certain defined conditions.
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

 Plate count method can be done in two ways either by spread plate method or by pour
plate method.
 This method of bacterial counting is most commonly used with satisfactory results for
the estimation of bacterial populations in milk, water, foods and many other
materials.
 This technique has some drawbacks because some relatively heat-sensitive
microorganisms may be damaged by the melted agar and will therefore be unable to
form colonies
Most Probable Number (MPN) Method
SERIAL DILUTIUON

 Another method for determining the number of bacteria in a sample is the most
probable number (MPN) method.
 This statistical estimating technique is based on the fact that the greater the number of
bacteria in a sample, the more dilution is needed to reduce the density to the point at
which no bacteria are left to grow in the tubes in a dilution series.
 The MPN method is most useful when the microbes being counted will not grow on
solid media (such as the chemoautotrophic nitrifying bacteria).
 It is also useful when the growth of bacteria in a liquid differential medium is used to
identify the microbes (such as coliform bacteria, which selectively ferment lactose to
acid, in water testing).
 The MPN is only a statement that there is a 95% chance that the bacterial population
falls within a certain range and that the MPN is statistically the most probable
number.

Using Turbidimetry:
A third method, known as turbidimetry, involves using a colorimeter to measure
the cloudiness or turbidity of the culture as cell numbers increase. Results are derived by
comparison with a standard graph of light absorbance plotted against known cell
numbers (You are not required to describe or use a calorimeter).  
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

DRY WEIGHT :
 For filamentous bacteria and molds, the usual measuring methods
are less satisfactory. A plate count would not measure this
increase in filamentous mass.
 In plate counts of actinomycetes and molds, it is mostly the number
of asexual spores that is counted instead.
 This is not a good measure of growth. One of the better ways to
measure the growth of filamentous organisms is by dry weight.
 In this procedure, the fungus is removed from the growth medium,
filtered to remove extraneous material, and dried in a desiccator, it
is then weighed.
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

 Growth measurement by measuring cell mass is one of the easiest

ways, a known volume of culture sample from the ferment or
withdrawn and centrifuged.
 It is the most direct approach for quantitative measurement of a
mass of cells.
Metabolic activity:
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

Microbial growth is defined as the increase in quantity of cellular constituents and structures
and is followed by an increase in size or number of cells or both. It is the division of the
microorganisms into two daughter cells. In the case of bacteria it is called binary fission. The
time of interval required for the division of microbial cell called generation time. The
population growth is studied by analyzing the growth curve of a microbial culture in different
systems.
SUMMARY:-
These are different system or mode of microbial cultures: 1. Closed System – Batch Culture
2. Fed Batch Culture 3. Continuous Culture (Open System) 4. Synchronous Culture.

1. Closed System – Batch Culture:

Microbial cells growing in a tube or flask of liquid medium are said to be in a closed system.
Here no nutrients are added to the system and no metabolic waste products are removed.
When nutrients are added to the system, the cells initially divide by binary fission and the cell
number increase for a period of time. Once the nutrients are over in the medium the growth
eventually stops and certain metabolic waste products are accumulated in the medium (Fig.
3.1).
A culture of microorganisms produced by inoculating a closed vessel containing a single
batch of medium is called a batch culture. Because no fresh medium is provided during
incubation, nutrient concentration decreases and concentration of waste products increases.
The growth of microorganisms reproducing by binary fission can be plotted on the logarithm
of the number of viable cells versus incubation time.
Fed Batch Culture:
In fed batch culture we add the growth limiting nutrient substrate to the culture. The fed batch
process is used in bio-industrial process to reach a high cell density in the bioreactor.
Controlled addition of the nutrient directly affects the growth rate of the culture and allows
avoiding overflow metabolism (formation of side metabolite such as acetate, lactic acid, etc.).
The substrate limitation offers the possibility to control the reaction rates to avoid
technological limitations connected to the cooling of the reactor and oxygen transfer.
Substrate limitation also allows the metabolic control, to avoid osmotic effects, catabolite
repression and metabolism of side products.
2. Continuous Culture (Open System):
Continuous culture system is a culture system with constant environmental condition
maintained through continuous supply of nutrients and removal of waste. Here, the cell
volume and cell concentration are kept constant by adding fresh, sterile medium. In industry,
it helps to maintain the active logarithmic growth phase which helps to generate maximum
Dr.Tulika Mishra, Deptt Of Botany, D.D.U G.U
BACTERIA

volume of desired products. The rate at which new cells are produced in the culture vessel is
balanced by the rate at which cell are being removed.
In continuous culture microbial population can be maintained in exponential growth phase
and at a constant biomass concentration for extended period
Two major types of continuous culture system are commonly used:
(1) Chemostat.
(2) Turbidostat.
1. Chemostat:
A chemostat (chemical environment is static) is a bioreactor to which fresh medium is
continuously added, while culture liquid is continuously removed to keep the volume of the
culture constant. The culture mediums for chemostat possess essential nutrients, (e.g.
amino acid) in limiting quantities.
Turbidostat:
It is the second type of continuous culture system which consists of a photocell that measures
the absorbance or turbidity of the culture in growth vessel. The flow rate of media through
the vessel is automatically regulated to maintain a predetermined turbidity on cell density.
The turbidostat differ from the chemostat –
1. The dilution rate in a turbidostat varies rather remaining constant, and its culture medium
contains all the nutrients.
The dilution rate is high, but in chemostat it is low.

3. Synchronous Culture:
Generally, all cells in a microbial culture do not divide at the same time. In a traditional batch
culture the microbial cells divided at random manner, i.e., the straight line that characterizes
the log phase of microbial culture in batch culture is due to random cell division. There are
certain laboratory techniques that manipulate the growth of cultures so that all cells divide at
the same time or grow synchronously.
Growth in a cell population in which all cells divide at same time is called synchronous
growth and such culture is called synchronous culture. A population can be synchronized by
changing the physical environment or chemical composition of the medium. If cells are
inoculated into a medium at a suboptimal temperature and kept that temperature, they will
metabolize slowly but will not divide.
When the temperature is rapidly increased at optimal level the cells undergo synchronised
division. Another method to obtain synchronised growth uses differential filtration or
centrifugation. When cells are separated by size, all cells are synchronised by each other. But
the synchronous culture lasts only a few generations.