B.Sc.

SEMESTER - IV
GENERAL MICROBIOLOGY
(Code: BBT - 401)
UNIT III
Microbial Growth: Growth curve, Generation time, synchronous, batch and continuous
culture, measurement of growth and factors affecting growth of bacteria.
Microbial Metabolism: Metabolic pathways, amphi-catabolic and biosynthetic pathways.
Bacterial Reproduction: Transformation, Transduction and Conjugation. Endospores and
sporulation in bacteria.
Synchronous Culture

• Synchronous cultures are composed of populations of cells that are physiologically

identical and in the same stage of cell division cycle at a given time.

• All the cells in the culture will divide at the same time will grow for a generation time,
and all will divide again at the same time.

• Thus the entire population is kept uniform with respect to growth and division.

• It is not possible to analyse a single bacterial cell to obtain the information about
growth behaviour, i.e. organisation, differentiation, and macro-molecular synthesis.

• Synchronous culture provides the entire cell crop in the same stage of growth.

• Measurement made on such cultures is equivalent to the measurement made on

individual cells.
Synchronous Culture Cont…

• In most of the bacterial cultures the stages of growth and cell division cycle are
completely random and thus it becomes difficult to understand the properties during
the course of division cycle using such cultures.

• To overcome this problem, the microbiologists have developed synchronous culture

techniques to find synchronous growth of bacterial population.

• A synchronous culture can be obtained either by manipulating environmental

conditions such as by repeatedly changing the temperature or by adding fresh nutrients
to cultures as soon as they enter the stationary phase, or by physical separation of cells
by centrifugation or filtration.
Synchronous Culture Cont…

• Two fundamentally different experimental approaches have been employed.

✦ In the first approach, a synchronous population of cells can be sorted out according
to age or size by physical separation of cells.

✦ In methods of second type, a culture is induced by manipulating the physical

environment or the chemical composition of the medium to obtain a synchronously
dividing population. The techniques based on selection are preferable to those
based on induction, since induction is likely to introduce distortions in the
physiologic state of the cells.
Synchronous Culture Cont…
1. Selection by Size and Age:
• A population of cells is fractionated on the basis of size.
• The cells are filtered so that smallest cells pass through the filter.
• These small cells are the youngest, and must go through their whole life-cycle before
dividing.

• Alternatively, the largest cells, which are ready to divide, may be retained or retarded
by a filter.

• These are then collected separately and used to obtain a synchronous culture.
• An excellent and most widely used method to obtain synchronous cultures is the
Helmstetter-Cummings Technique.
Helmstetter-Cummings Technique
• In this technique an unsynchronized bacterial culture is
filtered through cellulose nitrate membrane filter.

• The loosely bound bacterial cells are washed from the

filter, leaving some cells tightly associated with the filter.

• The filter is now inverted and fresh medium is allowed to

flow through it.

• New bacterial cells, that are produced by cell division and

are not tightly associated with the filter, are washed into
the effluent.

• Hence, all cells in the effluent are newly formed and are,
therefore at the same stage of growth and division cycle
(Fig. 19.4).

• The effluent thus represents a synchronous culture.

Synchronous Culture Cont…

2. Selection by Induction Technique:

• A synchronous culture is also obtained by the use of shock treatments.
• These include variation in temperature, starvation, exposure to light (for
photosynthetic organisms), drugs, and sub-lethal doses of radiation.
• A commonly used technique involves submitting a culture of microorganisms to single
or multiple changes in temperature.
• An exponentially growing culture at 37°C is held for about 30 minutes at 20°C. The
lower temperature retards cell division.
• During the interval of 30 minutes all the cells mature to the point of fission. However,
at 20°C none divide.
• On sudden return of the culture to 37°C, all the cells divide synchronously.
• By repeating the alterations of temperature, synchrony can be maintained in the culture
for several generations.
• Methods of inducing synchronous division based on changes in medium composition
have also been used.
2. Selection by Induction Technique Cont…

• A phasing of cell division is observed in cultures of a thymine-requiring mutant, following

withdrawal and re-addition of thymine to the culture medium.

• The time-course pattern of synthesis of various macromolecules in the cell cycle is studied by
removing portions of a synchronously dividing culture.

• The cells are then analysed for the content of macromolecules or enzyme activity.
• However, the optical density of the culture increases exponentially, since optical density
measures cells mass and mass is increasing.

• Similarly, the total synthesis of DNA, RNA, and protein increases exponentially.
Batch Culture
• A batch fermentation is regarded as a closed system.
• The sterile nutrient culture medium in the bioreactor is inoculated with microorganisms.
• The incubation is carried out under optimal physiological conditions (pH, temperature, O2
supply, agitation etc.).
• It may be necessary to add acid or alkali to maintain pH, and anti-foam agents to
minimise foam.
• Under optimal conditions for growth, the following six typical phases of growth are
observed in batch fermentation.
• 1. Lag phase
• 2. Acceleration phase
• 3. Logarithmic (log) phase (exponential phase)
• 4. Deceleration phase
• 5. Stationary phase
• 6. Death phase.
Batch Culture Cont…
1. Lag phase:
• The initial brief period of culturing after inoculation is referred to as lag phase.
• During the lag phase, the microorganisms adapt to the new environment—available
nutrients, pH etc.
• There is no increase in the cell number, although the cellular weight may slightly increase.
• The length of the lag phase is variable and is mostly determined by the new set of
physiological conditions, and the phase at which the microorganisms were existing when
inoculated.
• For instance, lag phase may not occur if the culture inoculated is at exponential phase (i.e.,
log phase), and growth may start immediately.
2. Acceleration phase: This is a brief transient period during which cells start growing
slowly. In fact, acceleration phase connects the lag phase and log phase.
Batch Culture Cont…

3. Log phase:
• The most active growth of microorganisms and multiplication occur during log phase.
• The cells undergo several doublings and the cell mass increases.
• When the number of cells or biomass is plotted against time on a semi logarithmic
graph, a straight line is obtained, hence the term log phase.
• Growth rate of microbes in log phase is independent of substrate (nutrient supply)
concentration as long as excess substrate is present, and there are no growth inhibitors
in the medium.
• In general, the specific growth rate of microorganisms for simpler substrates is greater
than for long chain molecules.
• This is explained on the basis of extra energy needed to split long chain substrates.

4. Deceleration phase: As the growth rate of microorganisms during log phase

decreases, they enter the deceleration phase. This phase is usually very short-lived and
may not be observable.
Batch Culture Cont…
5. Stationary phase:
• As the substrate in the growth medium gets depleted, and the metabolic end products
that are formed inhibit the growth, the cells enter the stationary phase.
• The microbial growth may either slow down or completely stop.
• The biomass may remain almost constant during stationary phase.
• This phase, however, is frequently associated with dramatic changes in the metabolism
of the cells which may produce compounds (secondary metabolites) of
biotechnological importance e.g. production of antibiotics.
6. Death phase:
• This phase is associated with cessation of metabolic activity and depletion of energy
reserves.
• The cells die at an exponential rate (a straight line may be obtained when the number
of surviving cells are plotted against time on a semi logarithmic plot).
• In the commercial and industrial fermentations, the growth of the microorganisms is
halted at the end of the log phase or just before the death phase begins, and the cells
are harvested.
Batch Culture Cont…

ADVANTAGES OF BATCH CULTURE:

1) Reduced risk of infection or cell mutation because the growth period is short.
2) Lower capital funding when in comparison to continuous processes for the same
bioreactor quantity.
3) More flexibility with varying product/organic structures.
4) Higher raw material conversion levels because of controlled growth rate.
DISADVANTAGES OF BATCH CULTURE:
1) Lower productivity stages due to time for filling, heating, sterilization, cooling,
emptying and cleaning the reactor.
2) Increased consciousness on instrumentation due to frequent sterilization.
3) More expense in preparing several subcultures for inoculation.
4) More investment for labour and process
5) More hygiene risks because of potential contact with pathogenic microorganisms or
pollution.
Continuous Culture
• It is possible to grow microorganisms in an open system, a system with constant
environmental conditions maintained through continual provision of nutrients and
removal of wastes.

• These conditions are met in the laboratory by a continuous culture system.

• A microbial population can be maintained in the exponential growth phase and at a

constant biomass concentration for extended periods in a continuous culture system.

• Two major types of continuous culture systems commonly are used:

✦ (1) chemostats and

✦(2) turbidostats
The Chemostat

• A chemostat is constructed so that sterile

medium is fed into the culture vessel at the
same rate as the media containing
microorganisms is removed (figure 6.9).

• The culture medium for a chemostat possesses

an essential nutrient (e.g., an amino acid) in
limiting quantities.

• Because of the presence of a limiting nutrient,

the growth rate is determined by the rate at
which new medium is fed into the growth
chamber, and the final cell density depends on
the concentration of the limiting nutrient.
Figure 6.9 A Continuous Culture System: The
Chemostat. Schematic diagram of the system.
The fresh medium contains a limiting amount of
an essential nutrient. Growth rate is determined
by the rate of flow of medium through the
culture vessel.
The Chemostat Cont…

• The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which
medium flows through the culture vessel relative to the vessel volume, where f is the
flow rate (ml/hr) and V is the vessel volume (ml).
D ︎= f/V

The Turbidostat
• The second type of continuous culture system, the turbidostat, has a photocell that
measures the absorbance or turbidity of the culture in the growth vessel.

• The flow rate of media through the vessel is automatically regulated to maintain a
predetermined turbidity or cell density.

• The turbidostat differs from the chemostat in several ways.

• The dilution rate in a turbidostat varies rather than remaining constant, and its culture
medium lacks a limiting nutrient.

• The turbidostat operates best at high dilution rates; the chemostat is most stable and
effective at lower dilution rates.
Schematic diagram of our turbidostat. It uses the
optical density measurements to modulate how much
media is pumped into the culture vessel every minute to
maintain a constant cell density. Through this method of
cell density control, we can measure how fast the cells are
growing and thus gain an understanding for relative
fitness increases thorough out the course of an
experiment.
Continuous Culture Cont…
• Continuous culture systems are very useful because they provide a constant supply of
cells in exponential phase and growing at a known rate.

• They make possible the study of microbial growth at very low nutrient levels,
concentrations close to those present in natural environments.

• These systems are essential for research in many areas—for example, in studies on
interactions between microbial species under environmental conditions resembling
those in a freshwater lake or pond.

• Continuous systems also are used in food and industrial microbiology.