Bacterial Growth

15 Hours

Rutam Mulay
Msc (Microbiology)

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 1

 Important Concepts
1. Understanding growth
2. The Growth Curve
3. Measurement of Bacterial Growth
4. Factors influencing Bacterial Growth
5. Diauxic growth
6. Synchronous growth
7. Microbial Energetics
8. Respiration (Aerobic, Anaerobic, Fermentation)

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 2

 WHAT IS GROWTH?

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 3

 Understanding Bacterial Growth!
Growth can be defined in terms of:
1. Increase in cellular constituents (cell size)
2. Increase in number.
Bacterial growth refers to an increase in
bacterial numbers, not an increase in the size of
the individual cells.
It is impractical to study microbial growth of
individual microorganisms due to their size.
Thus, total population number is used as a
standard to study microbial growth.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 4
 CONCEPT OF
GENERATION TIME

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 5

 Understanding Generation time!
The time required for a cell to divide and it’s
population to double is called the generation time.
It varies considerably among organisms and with
environmental conditions, such as temperature.
Most bacteria have a generation time of 1 to 3 hours;
others require more than 24 hours per generation.
In other words, one cell's division produces two cells,
two cells' divisions produce four cells, and so on.
When the no of cells in each generation is expressed
as a power of 2, the exponent tells the number of
doublings (generations) that have occurred.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 6
 HOW WOULD YOU
EXPRESS BACTERIAL
CELL COUNT?

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 7

 Using Numbers? Bad Idea…

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 8

 Logarithmic Representation!
It is important to understand the difference
between arithmetic and logarithmic
representation of cells.
Let’s consider 20 cell divisions in a bacterial
cell; after 5 generations, the total cell count will
be 32 and after 10 generations it will be 1042 and
so on.
Q. How do we represent this on the graph
paper?
Here the importance of log can be appreciated.
Don’t worry you can use calculator!!

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 9

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 10
 Arithmetic v/s Logarithmic

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 11

 Major reasons for using Log
values
1. Clear population changes can be
shown.
2. Large microbial population can be
represented in little space.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 12

 THE BACTERIAL GROWTH
CURVE

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 13

 Phases of bacterial growth
Microbial population is studied by analyzing the
growth curve of a microorganism only in a batch
culture system (closed system).
When a few bacterial cells are inoculated in the
broth, they start to grow.
The population can be counted at regular intervals
and results in a curve which is called as the growth
curve that represents the life span of the microbial
cell.
This growth of microorganisms reproducing by
binary fission can be plotted as the logarithm of
number
29-12-2019
of viable cells v/s incubation time.
F.Y.B.Sc Sem II_ Microbial Growth 14
 Phases of growth curve
1. LAG phase
2. LOG phase (exponential phase)
3. STATIONARY phase
4. DEATH phase (Phase of decline)

GRAPH
X axis – Time
Y axis – Log number of viable cells
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 15
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 16
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 17
1. Lag Phase
 No immediate increase in cell number, mass and lasts
for 1 hour to several days.
 Required for synthesis of ATP and growth factors.
 Required for synthesis of enzymes if the medium is
changed.
 Required for recovery of injured microorganisms.
 Key enzymes for replication of DNA and protein
synthesis are made.
 Varies depending on the type of organism.
 Lag phases are longer if inoculum is old or transferred
into a new medium; and shorter for young vigorously
growing organisms.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 18
2. Log phase (Exponential phase)
MO’s are growing at maximum possible rate.
The rate of growth is constant and the organisms are
dividing and doubling at regular intervals.
The population is uniform in terms of chemical and
physiological properties. These cultures are used in
biochemical, physiological and industrial microbiology.
Exponential growth is balanced growth.
Under unusual circumstances, unbalanced growth
results – Shift up and Shift down.
Log phase growth is not an indefinite growth as the
transport systems and enzymes get saturated.
The graph rises and then begins to plateau.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 19
3. Stationary phase (Levelling phase)
Growth ceases and graph becomes horizontal.
Attained by bacteria at a population level of around
10^9 cells per ml.
The microbial population ceases to divide but remains
metabolically active.
Reasons for entering stationary phase:
i. Nutrient limitation
ii. Accumulation of toxic waste products
 Starvation may be positive for some bacteria. They
produce starvation proteins (Dps) that protect the
cell.
Eg: S.typhi becomes more virulent when starved.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 20
4. Death phase
 Decline of viable cells post stationary phase.
 Death of organisms is exponential like log phase.
“Irreversible harm and loss of viability”.
This view is under debate with two hypotheses:
i. Viable but not culturable cells (VBNC)
ii. Programmed cell death (Apoptosis)
 Death is apparently good!
 Long term experiments reveal that there is a gradual
decline in culturable cells.
 This is because cells from same subpopulation are
able to tolerate the accumulated toxins (natural
selection).
F.Y.B.Sc Sem II_ Microbial Growth 21
29-12-2019
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 22
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 23
 MEASUREMENT OF
BACTERIAL GROWTH

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 24

 Basic Understanding…
•The growth of microbial populations can be measured
in a number of ways.
•Population numbers are usually recorded as the number
of cells in 1 ml of liquid or in 1 gram of solid material.
•Bacterial populations are usually very large, so all the
enumeration methods employ small sample volumes.
•Back calculations then determine the size of the total
population in the total volume of the sample.
•However it is impractical to divide 1g or 1ml of food in
small parts for analysis thus enumerations are done
indirectly by a process called serial dilutions.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 25
 Serial Dilution Technique
oLets suppose you have 10ml milk that contains
100,000 bacterial cells. Thus, 10,000 cells/ml.
oNow, can you count 10,000 colonies on one Petri
plate?
oSo what we do, we transfer 1ml of milk sample in 9ml
of sterile saline.
oNow how many cells would be present in the diluted
sample? Keep doing this step till you get a countable
plate.
oImportant – As you transfer 1ml to 9ml you are
diluting the sample 10 times. Dilution is represented
as 1:10 and dilution factor is 10.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 26
 Methods of Measurement
•Microbial growth can be measured by two ways:
1. Direct measurement
a. Plate counts
b. Filtration
c. Most probable number (MPN)
d. Direct microscopic count (DMC)
2. Indirect measurement
a. Turbidity
b. Metabolic activity
c. Dry weight
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 27
 Direct Methods
1. PLATE COUNTS (cfu/ml)
•Most common method used in all laboratories
involving all the samples.
•Major advantage – provides a count of viable cells.
•This method assumes that each viable bacterium
grows and divides to produce a single colony.
•It is practical to count colonies only when there are
limited number of colonies growing on the plate.
•US-FDA recommends to select plates with 25-250
colonies for enumeration.
•Serial dilutions are carried out for accuracy in counts.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 28
 Types of Plate Count Methods
•There are two major ways of enumerating bacterial cells
by plate count methods.
1. Surface spread method
2. Pour plate method
 These two methods provide the count for viable cells
only hence they are called viable count methods.
 Requirements for both the methods:
o Unknown sample
o St. agar plates or butts | St. Saline
o Dilution tubes, 1ml and 10ml pipettes, Petri plates
o Incubator at 37OC for 24 hours , spreader, alcohol
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 29
 Surface Spread v/s Pour plate
Particulars Spread plate method Pour plate method
Alternate names Surface seeding method Bulk seeding method

First to be added Agar in the plate Sample in empty plate

Second to be Sample on agar plate Molten agar (agar butt)
added
Volume of sample 0.1ml 1ml
Type of growth Surface growth Surface and submerged
growth
Organisms Aerobic, Aerobic, facultative anaerobic
microaerophilic
Diagnostic Yes No
application
Application Detection of E.coli from Total microbial count of soil or
food or water water
Accuracy Low High
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 30
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 31
 Surface Spread Method
Advantages
1. Heat sensitive microbes are not affected.
2. Isolation of the organism is easy.
Limitations
1. Only strict aerobes are enumerated.
2. 0.1 ml is used only (no greater volumes can be used).
3. Crowding can make counting difficult (dilution)
Applications
1. Cultural characteristics of the bacteria can be
studied.
2. Viable counts in various specimens.
3. Detection of pathogens from various samples.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 32
 Pour Plate Method
Advantages
1. Easy and gives accurate results (in terms of volume).
2. Does not require prepared and dried agar plates.
Limitations
1. Heat sensitive organisms may get killed.
2. Some colonies can be overlooked.
3. Contamination is difficult to identify.
Applications
1. Total viable count in a sample.
2. Enumeration of organisms from various samples.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 33

 Membrane Filtration
Can be used when the number of bacteria is less (eg:
in pure streams, drinking water etc).
Minimum amount of water that needs to be filtered is
100 ml. (sample volume depends on method used).
Apparatus consists of the following:
1. Filtration assembly
2. Membrane filter (0.45um, CA, CE, MCE)
3. Vacuum pump
4. Medium (selective depending on the requirement)
5. Forcep and alcohol

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 34

 Membrane Filtration Contd…
Protocol:
1. Mix the sample thoroughly.
2. Connect the MF assembly to vacuum source.
3. Pour the sample on the MF assembly.
4. Wait till all the sample is added.
5. Switch off the vacuum pump and pick up the
filter.
6. Place it on the media.
7. Incubate and count colonies present in the
sample.
8. Report as cfu/ml.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 35
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 36
 Direct Microscopic Count
A measured volume of a bacterial suspension is
placed within a defined area on a microscope slide.
A 0.01 ml sample is spread over a marked area of
slide, stain is added so that the bacteria can be seen,
and the sample is viewed under the oil immersion
objective lens.
Once the number of bacteria has been counted in
several different fields, the average number of
bacteria per viewing field can be calculated.
•The final count is given by multiplying the no of
cells with MF as cells/ml of the sample.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 37
 Applications
Count the number of bacteria present in the milk.
Rapid platform test.
Protocol same as for DMC.
Wilson’s chart helps to know how many fields need to
be counted. Avg No of bacteria/field No of fields to be counted
0-3 64
4-6 32
7-12 16
13-25 8
26-50 4
51-100 2
>100 1

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 38

 DMC
Advantages
1. Quick results.
2. No incubation required.
Limitations
1. High concentration of cells is preferred.
2. Difficult to count motile bacteria.
3. Live and dead cells cannot be differentiated.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 39

 Hemocytometer
•It is an counting chamber originally designed for
counting blood cells but now is used for cells in liquids.
•It is a thick glass microscopic slide with a rectangular
indentation that creates a chamber that has a grid of
perpendicular lines.
•It has a uniform depth of 0.1mm with dimensions of
3mm X 3mm.
•The total area of 9sq.mm is divided into 9 squares of
equal area (1sq.mm).
•Of the total 9 squares, we are only interested in the 5
squares.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 40
•The 4 squares placed at four corners are called WBC
squares while the central square is called RBC square.
•Each of the WBC square is further divided into 16
small squares.
•The central RBC square is divided into 25 squares
equal squares.
•Each of the 25 squares is further divided into 16
squares.
•Dense suspensions are c0unted in RBC squares
while less dense in WBC squares.
•Cell count in WBC squares = cells X 2500 / ml.
•Cell count in RBC squares = cells X 50,000 / ml.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 41
 Applications
1. Blood cell count
2. Sperm count
3. Brewing (yeast count)
4. Counting yeasts, cysts
5. Live and dead cell count (Trypan Blue staining)

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 44

 Most Probable Number
•The MPN method is most useful when the microbes
being counted will not grow on solid media.
•It is a method used to estimate the concentration of
viable MO’s in a sample by means of replicate liquid
broth growth in ten -fold dilutions.
•MPN is most commonly applied for quality testing of
water i.e to ensure whether the water is safe or not in
terms of bacteria present in it.
•Water potability is conducted in three steps:
• Presumptive test (MPN)
• Confirmed test
• Completed test
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 45
 Principle
•Water to be tested is diluted serially and inoculated in
lactose broth (single and double strength).
•Coliforms if present in water utilize the lactose
present in the medium to produce acid and gas.
•The presence of acid is indicated by colour change of
the medium and the presence of gas is detected as gas
bubbles collected in the inverted Durham tube.
•The number of total coliforms is determined by
counting the number of tubes giving positive
reaction (i.e both colour change and gas production).
•The pattern of positive results (the number of tubes
showing growth at each dilution) is compared with
standard statistical tables (95% confidence).
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 46
 INDIRECT METHODS

1. Turbidity
2. Metabolic activity
3. Dry weight
 Turbidity Measurements
•The basis of this method lies in the fact that microbial
cells scatter light striking them.
•As bacteria grow in the medium, the medium becomes
turbid or cloudy.
•The amount of scattering is directly proportional to
the biomass of cells present as microbes are of same
size.
•Spectrophotometer or colorimeter is used to measure
the light scattered (turbidity).
•When the concentration of bacteria reaches about 10
million cells per ml, the medium appears slightly
cloudy or turbid.
 Basic Principle
•When the light strikes bacterial cells, they will absorb
certain wavelength of light and transmit some light.
•If the turbidity is high, more light will be absorbed
and less light will be transmitted towards the detector.
•This reading will be noted as % transmittance.
•Also the instrument has another log function called
Abs (absorbance) or O.D calculated as Abs = 2-log %T.
•This absorbance value (direct read out) is relative of
the bacterial numbers which can be confirmed by
viable counts.
•Major limitation – 10 to 100 million cells are required
for such measurements.
 Metabolic Activity
•Assumption – the amount of some metabolic product
is directly related to bacterial growth.
•If the amount of a substance in each cell is constant,
the total quantity of that cell constituent is directly
related to the total microbial cell mass.
•Examples
1. Total protein content (microbial cells)
2. Chlorophyll content (photosynthetic bacteria)
3. ATP measurement (living cells)
 Dry Weight
•It is comparatively simple and can be used for fungal
cells (filamentous organisms).
•Simply, the fungal culture is grown in broth, the
hyphae are separated (filtration), dried in oven and
weighed on electronic balance.
•It is time consuming, however, and not very sensitive.
•Bacteria weigh so little that it is necessary to centrifuge
several hundred millilitres of culture to collect a
sufficient quantity of sediment.
•This method is not a very accurate method and lacks
sensitivity.
FACTORS INFLUENCING
MICROBIAL GROWTH
 Basic Understanding…
•Natural environments contain varying concentration of
nutrients (in a way it is a stressful environment for MO’s).
•Microbes are present virtually everywhere on Earth.
•Many habitats in which microbes thrive would kill most
other organisms.
•Bacteria such as Bacillus infernus are able to live over 2.4
kilometers below Earth’s surface, without oxygen and at
temperatures above 60°C.
•Microorganisms that grow in such harsh conditions are
called extremophiles.
•This topic helps in understanding ways to control microbial
growth and study ecological distribution of microorganisms.
 pH
•pH is a measure of the relative acidity of a solution
and is defined as the negative logarithm of the
hydrogen ion concentration.
•Each species has a definite pH growth range and pH
growth optimum.
•Acidophiles [pH 0 and 5.5], neutrophiles [pH 5.5 and
8.0]; and alkalophiles, [pH 8.0 and 11.5] & extreme
alkalophiles [>pH 10].
•Eg: Archaea are acidophiles, Bacteria are neutrophiles
and alkalophiles (Vibrio spp).
 Effect of pH on Microbial cells
When the external pH is low, the concentration of H
is greater outside than inside, and H+ will move into
the cytoplasm.
Drastic variations in cytoplasmic pH can harm
microbes by disrupting the plasma membrane or
inhibiting the activity of enzymes and membrane
transport proteins.
 Most prokaryotes die if the internal pH drops much
below 5.0 to 5.5.
Changes in the external pH also might alter the
ionization of nutrient molecules and thus reduce their
availability to the organism.
 Microbial Strategies
MO’s employ several mechanisms to maintain their
cytoplasmic pH at 7.0 irrespective of their classification.
1. Potassium antiport system.
2. Acid tolerance response.
• Proton translocating ATPase (pH 5.5-6.0) will make
ATP’s or pump H out of the cell.
• Acid shock proteins (pH 4.5) help in renaturation of
the misfolded proteins.
3. Extremophiles like Bacillus alcalophilus exchange Na
for external protons through exchange pumps.
SOLUTES AND WATER
ACTIVITY
Plasma membrane is selectively permeable and acts
as a physical barrier for entry of substances.
Challenge: If the organism is placed in the hypotonic
solution, water enters cell causing swelling and burst.
Solution: Inclusion bodies and pressure sensitive
channels that allow solute to escape when Oe>Oi.
Challenge: If the organism is placed in the hypertonic
solution, water leaves the cell causing plasmolysis.
Solution: Compatible solutes. Prokaryotes increase CS
production in hypertonic environment.
Examples of CS: choline, betaine, proline, glutamic
acid, polyols (arabitol, mannitol, glycerol), amino acids.
Solution 2: binding of water molecules by solutes
(osmotic effect) or solid surfaces (matric effect).
•The water available to the bacteria is significant and is
expressed in terms of water activity.
•Water activity is inversely proportional to osmotic
pressure.
•It is 1/100 of the relative humidity of the solution.
•It is given by the following formula:
 Halophiles – Salt Loving
•Adapted to hypertonic conditions and require high
salt concentrations (2.8 to 6 M) to grow.
•Solution:-
1. Compatible solutes
2. Modified enzymes, proteins (require high K)
3. Plasma membrane stabilization by high Na.
Example: Archaeon Halobacterium is isolated from the
Dead sea (salt lake between Israel and Jordan)
(34% salinity)
 OXYGEN
CONCENTRATION
Classification of organisms on oxygen requirement:
Type Oxygen requirement Example
Obligate aerobes Obligate requirement Micrococcus,
Pseudomonas,

Facultative anaerobes Do not require but grow Escherichia, Enterococcus

well in its presence

Aerotolerant anaerobes Ignore and grow well if it is Streptococcus pyogenes

present or absent

Obligate anaerobes Do not tolerate oxygen at Clostridium,

all Methanobacterium

Microaerophiles 2-10 % oxygen Campylobacter, Treponema

pallidum
 Oxygen and Bacterial Growth
•Bacteria can have more than one type of growth pattern;
fungi are aerobic and yeast are facultative anaerobes.
•Such environments provide flexibility and ecological
advantage to some organisms.
Eg: Strict anaerobe Bacteroides gingivalis lives in the
mouth where it grows in anaerobic crevices around teeth.
•The basic route taken by oxygen in metabolism results in
the formation of toxic reduced compounds viz:-
1. Superoxide radical
2. Hydrogen peroxide
3. Hydroxide radical
 Oxygen and Toxic Products
•These products are powerful oxidizing agents and are
extremely toxic resulting in cell death.
•Neutrophils and macrophages use these toxic oxygen
products to destroy invading pathogens.
 Strategies
•MO’s use a number of enzymes to detoxify these
toxic compounds ensuring their survival.
•These enzymes are:-
1. Superoxide dismutase
2. Catalase
3. Peroxidase
TEMPERATURE
 Basic Understanding…
•Environmental temperature profoundly affects MO’s,
especially their enzymes.
•Low temperature slows down growth while high
temperature destroys the cells.
•Enzyme activity roughly doubles for every 10°C rise in
temperature.
•Microbes have a distinct cardinal temperature range:-
minimum, maximum and optimum (near maximum).
•Organisms are known to grow from as low as -10 degrees
to as high as 100 degrees.
•Eukaryotes however cannot grow at temperatures above
60 degrees.
Classification Based on Temperature requirement:
Type Temperature REMARKS
requirement
Psychrophiles 0 degrees Plasma membrane has
(Pseudomonas, Vibrio, high levels of
Shwanella, Bacillus) unsaturated fatty acids.
0-7 degrees but can grow Similar mechanism and
Psychrotrophs up to 30 degrees responsible for spoilage
of refrigerated foods.
Mesophiles 20-45 degrees Almost most humans
(Salmonella, Shigella, (optimum of 37 degrees) are mesophiles.
Klebsiella)
More heat stable
Thermophiles 55 degrees and higher. enzymes and saturated
(Bacillus spp) FA in plasma
membranes.
Hyperthermophiles
(Pyrococcus abyssi, 90 degrees and higher Mechanisms not known
Pyrodictium occultum)
RADIATION
 Basic Understanding…
•The world is bombarded with radiation travelling like
waves with distance between crest and trough being
wavelength.
•As the wavelength of electromagnetic radiation
decreases, the energy of the radiation increases.
•Eg: gamma rays and X rays are much more energetic than
visible light or infrared waves.
•Sunlight is the major source of light and includes UV
radiation, IR radiation, radio waves.
•Many forms of radiation are harmful to humans and MO’s
including the ionizing radiations which have high energy
and short wavelength.
 Ionizing Radiation…
•Two major forms of ionizing radiation are (1) X rays,
which are artificially produced, and (2) gamma rays, which
are emitted during radioisotope decay.
•Low levels of ionizing radiation produces mutations and
resulting in death, while higher levels are directly lethal.
•Some procaryotes (e.g., Deinococcus radiodurans) and
bacterial endospores can survive ionizing radiation.
•Mechanisms of damage:-
1. Breakage of hydrogen bonds
2. Ring structure destruction
3. Oxidation of double bonds
4. Generation of hydroxyl radicals (in presence of oxygen)
 UV Radiation…
•It kills all types of organisms due to its short
wavelength and high energy (most lethal wavelength
is 260nm).
•Primary mechanism of damage at 260nm - formation
of thymine dimers in DNA.
• Repair mechanisms exist like photoreactivation, dark
repair, SOS repair.
•Primary mechanism of damage at 325-400nm –
breakdown of tryptophan into toxic end products.
•The exact mechanism is not known.
PRESSURE
 Barophiles…
•Normally, all MO’s experience 1 atm pressure which is
an ideal pressure for survival and growth.
•Organisms (barophiles) are known to survive in ocean
beds where the pressure is 600-1100 atm.
•Their mechanism of adaptation is not fully known to
us.
•Examples: Photobacterium, Shewanella, Colwellia
 Synchronous Growth

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 82

 What is synchronous growth?
Synchronous growth is the growth of bacteria such
that all the bacteria are at the same stage in their
growth cycle (e.g., exponential phase, stationary phase).
Synchronous growth permits the detection of events
not normally detectable in a single cell or in a
population consisting of bacteria in various stages of
growth.
In a normal batch culture of fluid, or on an agar plate,
bacteria in the population exhibit a range of sizes, ages,
and growth rates.
In contrast, the bacteria in a synchronized culture are
virtually identical in terms of these parameters.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 83
 Features of Synchronous Growth

1. Not shown by all bacteria.

2. Temporary (short period).
3. Important for Industrial Microbiology.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 84

 How to achieve synchronous growth?
1. Membrane Filtration
2. Centrifugation (density based methods)
3. Manipulation of environmental factor
(auxotrophs)
4. Addition of an antibiotic

 It is possible only for a few generations after

which inherent randomness dominates.
 For those few generations, however, much
useful information can be extracted from a
synchronously growing population.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 85
 CONTINUOUS CULTURING
OF MICROORGANISMS

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 86

 Why do we need Continuous
culturing systems?
1. Constant supply of cells in log phase.
2. Study growth at low nutrient concentrations
(mimic natural environments).
3. Food and industrial microbiology.
4. Microbial interactions.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 87

 Basic Understanding…
•Open system.
•Required environmental conditions maintained.
•Continuous supply of nutrients and removal of
waste products.
•This is again important for industrial
microbiology.
•Two ways of achieving continuous cultures:
1. Chemostat
2. Turbidostat
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 88
 Chemostat (BIOREACTOR)
•Sterile medium is fed into the culture vessel at the
same rate as the media containing microorganisms is
removed.
•The concentration of essential nutrient is kept in
limiting concentrations (eg: amino acid).
•The experimenter can maintain the steady state
growth.
•The growth rate is determined by the rate at which
new medium is fed in the system.
•This is called as dilution rate expressed as D=f(ml/hr)
/v (ml).
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 89
 Chemostat continued…
•If the dilution rate rises too high, the microorganisms can
be washed out of the culture vessel before reproducing
because the dilution rate >maximum growth rate.
• The limiting nutrient concentration rises at higher
dilution rates because fewer microorganisms are present
to use it.
•Only a limited supply of nutrient is available at low
dilution rates.
•The available energy is then used for cell maintenance,
not for growth and reproduction.
•As D increases, more nutrients are available so the growth
rate also increases.
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 90
29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 91
 Turbidostat…

•It is based on the turbidity measurement of the cell.

•It uses a photocell to measure the turbidity (the
instrument used is spectrophotometer).
•It is principally similar to chemostat but is slightly
different in terms of the flow rate (variable).
•This variable flow rate keeps the turbidity constant in
the medium.

29-12-2019 F.Y.B.Sc Sem II_ Microbial Growth 92