Study & plot the growth curve of E coli. using turbidimetric method and calculate the growth rate specificity, generation time, Lab Reports of Molecular biology

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STUDY AND PLOT THE GROWTH CURVE OF Escherichia coli USING TURBIDOMETRIC

METHOD AND CALCULATE GROWTH RATE AND GENERATION TIME.

AIM: To study and plot the growth curve of Escherichia coli using turbidometric

method and to calculate specific growth rate constant and generation time.

THEORY: Bacterial growth is characterized by an increase in number of

bacterial cells in the subsequent generation,not just by an increase in cell mass

and size.Most bateria replicates by binary fission,also known as amitosis(different

from eukaryotic mitosis) in which cell division occurs without the stages of a

typical mitosis.

Binary fission is relatively simple

wherein the chromosome is replicated and the mother cell is finaly divided into

two daughter cells by septum formation.Therefore, bacteria increases their

numbers by geometric progression whereby the population gets doubled in a fixed

period of time called generation time or doubling time.

Generation time is the time taken by a given number of bacteria to get

doubled.Although the bacteria are capable of replicating exponentially as a result

of Binary fission, in reality, this only occurs as long as there is ample space to grow,

sufficient nutrients and low concentration of toxic products released by the

preexisting cells.With the progression of growth,less nutrients and less space is

and high concentration of toxic products limit the ability to replicate exponentially

over time in a closed culture system,there is sharp decline and flattening of growth

rate.This entire scenario can be depicted as a curve known as growth curve that

consists of several stages :

1.Lag phase(region ‘a’ of the above curve ) : During this phase there is no

visible growth,and the curve is is relatively flat because in this phase the number

of cells do not increase.But that doesnot mean the cells are metabolically inactive.

aids (as nutrient broth solution turn kind of cloudy or turbid when bacterial cells

are present in the order 10

6

cells per ml )

It is of utmost importance what wavelength do we choose to

achieve our task. We usually use 600 nm wavelength. The cells do not actually

absorb EM radiation at 600 nm , rather they scatter it, as a result radiation of

lesser intensity reaches the detector implying less trasmittance so indirectly we

can say O.D value or absorbance is more. Thus, more the cell number(turbidity) or

biomass concentration lesser is transmittance and more O.D value and vice-versa.

Thus at regular intervals of incubation period if O.D is checked, it

increases as bacterial cell biomass concentration or number increases .Now, these

values can be used to plot an O.D 600

vs time curve which would well be a proper

representation of bacterial growth ;i.e a sigmoid curve.

After a short lag phase, the cells enter a phase of rapid exponential or logarithmic

phase , wherein rate of increase of biomass concentration is directly proportional

to initial biomass concentration.

𝑑𝑥

𝑑𝑡

∝x

Or,

𝑑𝑥

𝑑𝑡

= μx ………………………(1) where,

μ=specific growth rate constant.

Or,

𝑑𝑥/𝑑𝑡

𝑥

=μ

When x = 1

𝑑𝑥

𝑑𝑡

= μ

Thus , specific growth rate constant may be defined as the rate of increase of

biomass concentration per unit biomass. It’s unit is time

• 1

From equation 1

𝑑𝑥

𝑑𝑡

=μx

Or,

𝑑𝑥

𝑥

= μdt

Integrating both sides,

𝑑𝑥

𝑥

𝑥

𝑥 0

=μ ∫

𝑡

0

ln

𝑥

𝑥𝑜

=μt

or, ln x – ln x 0

=μt

or , ln x = ln x 0

+μt…………………..( 2)

or, x =x 0

e

μt

Now when t=t g

, x =2x 0 ,

therefore equation 2 becomes,

ln 2x 0

=ln x o

+μt g

or, ln 2 = μt g

or , t g

𝟎.𝟔𝟗𝟑

μ

or, μ=

𝟎.𝟔𝟗𝟑

𝒕𝒈

t g

or the generation time or the doubling time is the time in which bacterial

biomass gets doubled (for E.coli it’s approximately 20 to 30 min at 37°c )

REQUIREMENTS:

 Overnight grown E.coli culture

 Nutrient broth

 Microtip (1ml)

 Spectrophotometer

 Micropipette

 Autoclave

 70% alcohol

OBSERVATION:

TABLE 1

OD

600

in nutrient broth….

Time (minute) Nutrient broth OD

600

GRAPH:

In the underline graph the ‘X’ axis represents time in minutes while the ‘Y’

axis represents OD at 600 nm.

CALCULATION: Imputing the value in equation (5).