Bacteria: Growth Curve, Isolation and Preservation

Unit I
Pharmaceutical Microbiology
B. Pharm III Sem.

Bacteria divide by binary fission
Alternative means
Budding
Conidiospores (filamentous
bacteria)
Fragmentation
Bacterial Growth
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GurunanakCollegeofPharmacy,Nagpur

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 Time required for cell to divide/for population
to double
 Average for bacteria is 1-3 hours
 E. coli generation time = 20 min
 20 generations (7 hours), 1 cell becomes 1
million cells!
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Generation Time

Bacterial Growth Curve
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Phases of Bacterial Growth Curve
Phase I: Lag Phase
(Initial Adjustment Phase)
Phase II: Phase of increasing Growth Rate
(1st Transition Phase)
Phase III: Logarithmic Phase
(Exponential Growth Phase)
Phase IV: Phase of Decreasing Growth Rate
(2nd Transition Phase)
Phase V: Stationary Phase
Phase VI: Phase of Increasing Death Rate
(3rd Transition Phase)
Phase VII: Logarithmic Death Phase
(Exponential Death Phase)
Phase VIII: Survival Phase
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Phase I: Lag Phase
(Initial Adjustment Phase)
Phase in which there is no change in bacterial count in
initial 2-4 hours after inoculation is called as lag phase.
In this phase bacterial cell adjust with the new environment
and prepares new enzymes in response to new medium.
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Phase II: Phase of increasing Growth Rate
(1st Transition Phase)
 In this phase some bacterial cells starts multiplying,
hence there will be slight increase in bacterial count.
 At the end of this phase all cells will be in rapid
multiplication stage
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Phase III: Logarithmic Phase
(Exponential Growth Phase)
 In this phase bacterial cell is in most active condition.
 Nutrients are abundent and metabolites are very less
hence bacterial cells multiply in exponential manner.
 This phase is desired for production of microbial
products
 Bacterial cell is most sensitive to drugs and radiation
during this period
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Phase IV: Phase of Decreasing Growth Rate
(2nd Transition Phase)
At the end of Log Phase there is depletion of nutrients
and accumulation of toxic metabolites.
Due to this cells start to die and decline in viable
count takes place.
Since many cells are actively multiplying, sudden
decrease will not take place.
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 Phase V: Stationary Phase
 The "stationary phase" is due to a growth-limiting factor; this is
mostly depletion of a nutrient, and/or the formation of
inhibitory products such as organic acids.
 Stationary phase results from a situation in which growth rate
and death rate have the same values
 (newly formed cells per time = dying cells per time)
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 Phase VI: Phase of Increasing Death Rate
(3rd Transition Phase)
 At the end of stationary phase, proportion of dying
cells will increase than the no. of cells multiplying.
 This results in third transition phase: Phase of
increasing death rate.
 This is due to very much accumulation of toxic
compounds.
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Phase VII: Logarithmic Death Phase
(Exponential Death Phase)
 Bacteria run out of nutrients and die although number of cells
remain constant.
 The decline phase is brought by exhaution of nutrients,
accumulation of toxic products and autolytic enzymes.
 There will be exponential death occurring in this phase and
viable cell count will go on reducing drastically.
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Phase VIII: Survival Phase
 Sometimes a small numbers of survivors may persist for
month even after death of majority of cells these few surviving
cells probably grow at expence of nutrients released.
The reason behind this may be
 Mutation: At starving condition cells get mutated to take
available substances as their nutrient.
 Spore Formation: The bacterial cells get converted into their
dormant form i.e. Spores.
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Growth of bacteria is affected by many factors such as
 Nutrition concentration
 Temperature
 Gaseous concentration
 pH
 Ions and salt concentration
 Available water
Factors affecting Bacterial Growth
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 Diauxic growth is any cell growth characterized by
cellular growth in two phases
 This is illustrated with a diauxic growth curve
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Diauxic Phenomenon

 In this phenomenon the preferred sugar is consumed first,
which leads to rapid growth, followed by a lag phase.
 During the lag phase the cellular machinery used to
metabolize the second sugar is activated
 Subsequently the second sugar is metabolized
 Remaining steps will be same as that of bacterial growth
curve.
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Diauxic Phenomenon

 Bacteria grow nonsynchronously in
ordinary culture medium, i.e at any
moment cells are present in different
stage of growth cycle.
 When all bacterial cells in culture
medium divide simultaneously growth
thus obtained is known as synchronous
growth.
 Such growth is required for studing the
sequence of event occuring in single cell
like studies on DNA synthesis or
susceptibility of cell to lethal agent
Synchronous Bacterial Growth
Culture
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 External conditions can be changed, so as to arrest growth of
all cells in the culture, and then changed again to resume
growth. The newly growing cells are now all starting to grow at
the same stage, and they are synchronized. e.g.
for photosynthetic cells, light can be eliminated for several
hours and then re-introduced.
 Eliminate an essential nutrient from the growth medium and
later re-introduce it.[5]
 Cell growth can also be arrested using chemical growth
inhibitors.
e.g. Nocodazole, for example, has been used in biological
research for synchronization.
 Physical Separation based on their density or size, for
instance. This can be achieved using centrifugation (for
density) or filtration (for size).
Methods for Synchronous Bacterial
Growth
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Helmstetter-Cummings technique
In this technique, a bacterial culture is filtered through a
membrane. Most bacteria pass through, but some remain
bound to the membrane. Fresh medium is then applied to the
membrane and the bound bacteria start to grow. Newborn
bacteria that detach from the membrane are now all at the
same stage of growth; they are collected in a flask that now
harbors a synchronous culture
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Methods for Synchronous Bacterial
Growth

Continuous Bacterial Growth Culture
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Continuous bacterial growth culture aims to keep a
culture growing indefinitely.
In this the bacterial culture is maintained in the
exponential growth phase.
Continuous culture is important in industrial processes
that harvest the primary metabolites.
This can be achieved by:
Fresh nutrients are continually supplied
Accumulated cells and waste products are removed at
the same rate
Conditions such as temperature and pH are kept at their
optimum values

Methods for Continuous Bacterial
Growth
Chemostat System
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 A turbidostat dynamically
adjusts the flow rate (and
therefore the dilution rate) to
make the turbidity constant.
At steady state, operation of
both the chemostat and
turbidostat are identical.
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Continuous Bacterial Growth
Turbidostat System

 Pure culture : containing a single species of
organism.
 Isolation of Bacterial Pure Culture
 A pure culture is usually derived from a mixed
culture (one containing many species) by
transferring a small sample into new, sterile growth
medium in such a manner as to disperse the
individual cells across the medium surface or by
thinning the sample many times before inoculating
the new medium
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Isolation of Bacterial Pure Culture

 Importance of Isolation of Bacterial Pure Culture
Once purified, the isolated species can then be cultivated
with the knowledge that only the desired microorganism
is being grown.
A pure culture can be correctly identified for accurate
studying and testing, and diagnosis in a clinical
environment.
Testing/experimenting with a pure culture ensures that the
same results can be achieved regardless of how many
time the test is repeated.
Pure culture spontaneous mutation rate is low.
Pure culture clone is 99.999% identical.
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 Cultures composed of cells arising from a single progenitor
 Progenitor is termed a CFU (Colony Forming Unit)
 Aseptic technique prevents contamination of sterile
substances or objects
 Techniques For isolation
 Streak plate method
 Pour plate method
 Spread plate method
 Roll tube method
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 Special Methods of Isolation of Bacteria
 Single Cell Isolation
 Capillary Pipette Method
 Micromanipulator Method
 Enrichment Culture Method
 The enrichment culture strategy provides a
specially designed cultural environment by
incorporating a specific nutrient in the medium and
by modifying the physical conditions of the
incubator
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 Streak plate method
 Streaking is the process of spreading the microbial
culture with an inoculating needle on the surface of the
media.
 Sterilize the inoculating needle by flame to make red
hot and allow it to cool for 30 seconds.
 Thesample is streaked in such a way to provide series
of dilution.
 purpose-thin out innoculum to get seprate colonies.
 Subculturing can be done by streaking well isolated
colonies from streak plate to new plate.
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I
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Various Patterns of the streaking

Pour plate method
 The bacterial culture and liquid agar medium are
mixed together.
 After mixing the medium, the medium containing
the culture poured into sterilized
 Petri dishes ( Petri plates), allowed solidifying and
then incubated.
 After incubation colonies appear on the surface.
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 Disadvantages of pour plate technique:
 Microorganism trapped beneath the surface of medium
hence surface as well as subsurface Colonies are
developed which makes the difficulties in counting the
bacterial colony.
 Tedious and time consuming method, microbes are
subjected to heat shock because liquid
 Medium maintained at 45℃., hence is unsuitable for
psychrophile
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 Spread plate method
 This is the best method to isolate thepure colonies.
 In this technique, the culture is not mixed with the agar
medium. Instead it is mixed with
 normal saline and serially diluted.
 0.1 ml of sample taken from diluted mixture, which is
placed on the surface of the agar plate
 and spread evenly over the surface by using L shaped
glass rod called spreader.
 Incubate the plates
 After incubation, colonies are observed on the agar
surface.
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Advantages:
1. It is a simple method.
2. In this method only surface colonies are formed.
3. Micro-organisms are not exposed to higher temperature.

 Roll tube method
 2ml nutrient agar melted, cooled to 50 centigrade, with 0.02 ml
pipette one drop of culture is added to test tube.
 Tubes rolled in horizontal position under cold water tap to
make uniform agar layer, incubated and colonies counted.
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 Micromanipulator method
 Micromanipulators have been built, which permit
one to pick out a single cell from a mixed
 culture. This instrument is used in conjunction with
a microscope to pick a single cell
 (particularly bacterial cell) from a hanging drop
preparation.
 The single cell of microbe sucked into micropipette
and transferred to large amount of sterile medium.
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 ADVANTAGES OF MICROMANIPULATOR METHOD
 The advantages of this method are that one can be
reasonably sure that the cultures come
 from a single cell and one can obtain strains with in the
species.
 DISADVANTAGES
 The disadvantages are that the equipment is expensive, its
manipulation is very tedious, and it requires a skilled person.
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 Enrichment Culture Method
 The enrichment culture strategy provides a specially
designed cultural environment by incorporating a
specific nutrient in the medium and by modifying the
physical conditions of the incubator
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 Preservation
 To maintain pure culture for extended periods in a viable
conditions, without any genetic change is referred as
Preservation.
 The aim of preservation is to stop the cell division at a
particular stage i.e. to stop microbial growth or at least
lower the growth rate.
 Due to this toxic chemicals are not accumulated and
hence viability of microorganisms is not affected.
 Objectives of preservation
 To maintain isolated pure cultures for extended periods
in a viable conditions.
 To avoid the contamination.
 To restrict genetic change (Mutation)
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Preservation of Bacterial Pure Culture

 Method of Preservation
Short Term Methods
 Periodic transfer to fresh media (Subculturing)
 Preservation of bacteria using glycerol
 Storage by refrigeration
Long Term Methods
 Mineral oil or liquid paraffin storage
 Storage in saline suspension
 Storage in sterile soil
 Lyophilization (freeze–drying)
 Cryopreservation
 Stored in silica gel
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 Periodic transfer to fresh media (Subculturing)
 The bacterial culture can be stored for longer time by
addition of bacterial cells in fresh medium periodically
 Many of the more common microbes remain viable for
several weeks or months on a medium like Nutrient agar.
 It is an advantageous as it is a simple method and any
special apparatus are not required.
 It is easy to recover the culture.
 The transfer has the disadvantage of failing to prevent
changes in the characteristics of a strain due to
development of variants and mutants and risk of
contamination is also more in this process.
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Preservation of bacteria using glycerol
 Bacteria can be frozen using 15% glycerol.
 The glycerol is diluted to 30% and an equal amount of
glycerol and culture broth are mixed, dispensed into
tubes, and then frozen at -10˚ C.
 The viability of organisms varied such as Escherichia
coli, Diplococcus pneumonia etc. viable for 5 months,
Haemophilus influnzae viable for 4 months,
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 Storage by refrigeration
 Pure cultures can be successfully stored at 0-4°C either
in refrigerators or incold-rooms.
 At this temperature range the metabolic activities of
microbes slows down greatly and only small quantity of
nutrients will be utilized.
 This method is applied for short duration (2-3 weeks for
bacteria and 3-4months for fungi) because the metabolic
activities of the microorganisms are greatly slowed down
but not stopped.
 Thus their growth continue slowly, nutrients are utilized
and waste products released in medium.
 This results in finally the death of the microorganisms
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 Mineral oil or liquid paraffin storage
 In this method sterile liquid paraffin is
poured over the slant culture of
microbes and stored upright at room
temperature.
 Where as cultures can also be
maintained by covering agar slants by
sterile mineral oil which is stored at
room temperature or preferably at 0-
5°C.
 It limit the oxygen access that reduces
the microorganism’s metabolism and
growth, as well as to cell drying during
preservation.
 The preservation period for bacteria
from the genera Azotobacter and
Mycobacterium is from 7-10 years, for
Bacillus it is 8-12 years.
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 Storage in saline suspension
 Bacterial culture is preserved in 1% salt concentration in
screw caped tubes to prevent evaporation.
 The tubes are stored in room temperature.
 Whenever needed the transfer is made on Agar Slant.
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 Storage in sterile soil
 Soil storage involves inoculation of 1ml of spore
suspension into soil (autoclaved twice) and incubating at
room temperature for 5-10 days.
 The initial growth period allows the fungus to use the
available moisture and gradually to become dormant.
 The bottles are then stored at refrigerator.
 Viability of organisms found around 70- 80 years
 It is mainly applied for the preservation of sporulating
microorganisms
 Fusarium, Penicillium, Alternaria, Rhizopus, Bacillus,
Aspergillus, Penicillium, etc. proved successful for store
in sterile soil.
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 Lyophilization (freeze–drying)
 It is a vacuum sublimation technique.
 Freeze drying products are hygroscopic and must be protected
from moisture during storage.
 By freezing the cells in a medium that contain a lyoprotectant
(usually sucrose) and then pulling the water out using vacuum
(sublimation), cells can be effectively preserved.
 Freezing must be very rapid, with the temperature lowered to
well below 0˚C (as such -20˚C).
 Lyophilized cultures are stored in the dark 4˚C in refrigerators.
 Many microbes preserved by this method have remained
viable and unchanged in their characteristic more than 20
years.
 It is very advantageous as only minimal storage space is
required to preserve.
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For Details of Lyophilization Procedure
https://www.youtube.com/watch?v=_jmJ3i7g9TQ

 Procedure of Lyophilization
 In this process, a dense cell suspension is placed in
small vials and frozen at -60 to -70°C.
 The vial are immediately connected to a high vacuum
line.
 The ice present in the frozen suspension
evaporates(sublime) under the vacuum.
 This results in dehydration of bacterial cell and their
metabolic activities are stopped; as a result, the
microbes go into dormant state and retain viability for
years.
 The vials are then sealed off under a vacuum and stored
in the dark at 4°C in refrigerators.
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 Cryopreservation
 Cryopreservation (i.e., freezing in liquid nitrogen at -196°C
or in the gas phase above the liquid nitrogen at -150°C)
helps survival of pure cultures for long storage times.
 In this method, the microorganisms of culture are rapidly
frozen in liquid nitrogen at -196°C in the presence of
stabilizing agents such as glycerol or Dimethyl Sulfoxide
(DMSO) that prevent the cell damage due to formation of
ice crystals and promote cell survival.
 This liquid nitrogen method has been successful with many
species that cannot be preserved by lyophilization and
most species can remain viable under these conditions for
10 to 30 years without undergoing change in thei
characteristics, however this method is expensive.
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 Stored in silica gel
 Microbes can be stored in silica gel powder at low
temperature for a period 1- 2 years.
 The basic principle in this technique is quick desiccation
at low temperature, which allows the cell to remain
viable for a long period of time.
 Some of the species which are preserved on anhydrous
silica gel are such as Saccharomyces cerevisiae,
Aspergillus nidulans, Pseudomonas denitrificans,
Escherichia coli etc.
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Bacteria: Growth Curve, Isolation and Preservation

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