ABSTRACT

Microorganism or parasite are mainly organism that can only be detectable with the
aid of equipment called microscope, which designed for high magnifications. A process
called mitosis process is their reproduction that involves duplication of nucleus through cell
division of the cell. The objective of this experiment are to study/observe the growth of
kinetics of microorganism in shake flask experiment, to construct a growth curve including
lag, log, stationary and death phases and to determine the Monod parameters of maximum
growth rate (µmax), mass doubling time (td) and specific growth rate (µnet). Erlenmeyer flask is
used in this experiment to grow microorganisms. Escherichia coli (E.Coli) is grown in
Terrific Broth (TB) medium at 350 rpm and 37°C. They were fermented in incubator shaker
for 24 hours. Throughout every 2 hours, cell is taken out to obtain the value of absorbance by
using spectrophotometer. Cell dry is then obtained after the mass concentration inside the
flask is dried overnight. As for the optical density analysis, the absorbance reading from the
spectrophotometer is taken. The cell dry weight, in the other hand, is taken after the mass
concentration is being dried overnight in the oven. The weight of the tube which contains the
biomass before and after the drying process is recorded to get the cell dry weight. Then, all
obtained value are turned into a graph to observe the changes in growth kinetics of the cell.

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INTRODUCTION

Microorganism or parasite are mainly organism that can only be detectable with the
aid of equipment called microscope, which designed for high magnifications. The nature of
the microorganisms are algae, bacteria, moulds, protozoa, viruses and yeast are normally
considered by scientists to as a non-living entity.
Microorganisms can be present everywhere in our environment like air we breathe,
soil, water, various parts of our bodies and the food that we take. A process called mitosis
process is their reproduction that involves duplication of nucleus through cell division of the
cell. Reproduction of microorganisms can also occur when a haploid nuclei unit to form a
diploid cell, changing further to bring about sexually produced offspring.
Microorganisms are like any other living things that needs a favourable circumstances
for growth, which can be slightly varied with microorganisms. Some of these circumstances
commonly considered as factors that influencing the growth of these entities. The factors that
can influenced the growth of microorganisms are pH, nutrition, moisture, oxygen,
temperature and time.
Bacterial population growth studies require inoculation of viable cells into a sterile
broth medium and incubation of the culture under optimum temperature, pH, and gaseous
conditions. Under these conditions, the cells will reproduce rapidly and the dynamics of the
microbial growth can be charted by means of a population growth curve, which is constructed
by plotting the increase in cell numbers versus time of incubation and can be used to
delineate stages of the growth cycle. It also facilitates measurement of cell numbers and the
rate of growth of a particular organism under standardized conditions as expressed by its
generation time, the time required for a microbial population to double.
There are many ways to carried out Fermentation process such as batch, continuous
and fed-batch processes. In this experiment, we decided to use the shake flask fermentation.
The shake flask fermentation is an example of batch fermentation. In shake flask, the culture
flask usually Erlenmeyer flask is being used to place and growing the microorganisms. It is a
small scale equipment which equivalent to stirred tank bioreactor and it is also the cheapest
and easiest way to culture microorganism aerobically, in small volumes of nutrient broth. 
Usually, complex media are used for shake flask cultures. However, to enhance the
growing the synthetic medium is being devised for the fermentation process. Studies on
inoculum size, temperature, agitation, nutrition are initially done using these cultures to
monitor their influences on growth and product formation.

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 There are many specific media for certain microorganisms like Luria Bertani
(Lennox) and Terrific Broth media. Bacterial E.coli growth media: LB Miller broth/LB
Lennox broth is the most commonly used medium in molecular biology for E.coli cell
culture. LB broth contains the enzymatic digestion product of casein commonly known as
peptone (some vendors term it Tryptone), yeast extract, and sodium chloride. Peptone is rich
in amino acids and peptides. Its amino acid and peptide compositions reflect those of casein.
In addition to amino acids and peptides, yeast extract also contains nucleic acids, lipids and
other nutrients which are needed for bacterial growth. (LB Miller, Lennox).
Other media is Bacterial E. coli growth medium TB or Terrific Broth TB or Terrific
broth is a phosphate buffered rich medium. In addition to 20% more peptone and 380% more
yeast extract than LB broth, TB also has an added 0.4% glycerol as an extra carbon source.
All these nutrients in TB can support E. coli growth to OD600 5 to 8 under normal shaking
incubation conditions. TB is commonly used for protein expression and plasmid production
in a laboratory scale.

OBJECTIVE
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In this experiment, the objectives were to:
 To study/observe the growth of kinetics of microorganism in shake flask experiment.
 To construct a growth curve including lag, log, stationary and death phases.
 To determine the Monod parameters of maximum growth rate (µ max), mass doubling
time (td) and specific growth rate (µnet).

THEORY

Lag phase is the time between inoculation and reaching the maximum growth rate.
There are two sub phases in the lag phase. In the first phase, there is no growth identified
whereas in the second sub phase which is also known as acceleration phase, there is a
constant growth begins. At the first phase, which is lagging phase where the cells are put into
the new medium, they will first go through in a long time to adapt to the new medium. The
cells are generating the enzymes they need to adapt and reproduce in the new medium.
The second phase is exponential phase also known as log phase. The cells begin to
proliferate with their maximum growth rate. The doubling time of E.coli is 20 minutes.
Exponential phase is important for determining the maximum growth rate, μ and doubling
time, d since the growth at this time is the most constant and ideal. At this phase, the cells are
actively dividing. At the same time, the nutrients supplied is reduced, and the waste products
from the cells build up increase. So, the pH may be changed by metabolic waste products.
Retardation phase is the period between exponential and stationary phase, or in other
words, the phase before the growth becomes stationary. Among the factors that inhibit the
growth are reduced dissolved oxygen tension (DOT), substrate concentration, pH changes
and presence of inhibiting metabolites.
After retardation phase, the growth phase enters stationary phase where the growth
becomes constant for a period of time before it declines. During this stage, the number of
cells undergoing division is equal to the number of cells that are dying. There is no further
increase in cell number and the population is maintained at its maximum level for a period of
time. The primary factors responsible for this phase are the depletion of some essential
metabolites and the accumulation of toxic acidic or alkaline end-products in the medium.
Finally, the growth declines from its stationary phase due to the cells lysation. This is
indicated by the decrease of the viable cell number. Because of the continuing depletion of
nutrients and buildup of metabolic wastes, the microorganisms die at a rapid and uniform
rate.

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This decrease in population closely parallels its increase during the log phase. Theoretically,
the entire population should die during a time interval equal to that of the log phase. This
does not occur, however, since a small number of highly resistant organisms persist for an
indeterminate length of time.

Calculation that involved in this experiment in order to achieve the stated objective:

 Rate of microbial growth μnet is characterised by specific growth rate:

1 dX 1
μnet≅ [ ]
X dt h
= gradient of the graph ln x versus time

 Mass doubling time (τ d) is calculated based on cell number and the net specific rate of
replication.

ln2
τ d= [h]
μmax

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APPARATUS AND REAGENT

1. Microbe: Escherichia Coli (E coli)

2.  Shake flask (250mL flasks and 1000 mL flasks)
3. Eppendorf tubes / falcon tube (1.5 mL)
4.  Cuvettes (spectrophotometer)
5.  Incubator Shaker
6. Refrigerated Centrifuge
7.  Media (for specific microbe)
8. Ethanol (70% ethanol for swabbing for sterility)
9. Spectrophotometer
10. Bunsen burner for sterility
11. Graduated Flask for measuring media (1000mL, 100mL, 10mL)
12. Laminar Flow hood for sterility
13.  Biochemical Analyzer
14.  HPLC for product measurement like ethanol
15. Cotton plugged
16. pH meter

6
PROCEDURE

(i) Preparation of media

Media must be prepared according to the needs of the microorganisms used.

Microorganisms used is Escherichia coli (E coli). The media used in this experiment is
Terrific Broth (TB). Terrific Broth is a readied phosphate buffer media. The chosen
media were prepared according to the recommended formula or recipe stated at the
chemical bottle.

No Name of media Brand Recipe (g/L)

1 Terrific Broth (TB) SRL Chem or 47.60 g to be filled
Merck up to 1L volume
Glycerol as carbon
source (4mL/1L)

a) Terrific Broth (TB) preparation (was prepared by lab technician).

1. The recipe as stated at the bottle is followed.

2. The media is autoclaved at 121°C for 20 minutes.
3. Glycerol and media has been autoclaved together.
4. pH reading should be near 7 as the media is a readied phosphate buffer solution.

(ii) Preparation of cell culture

a) Seed culture preparation (inoculums)

1. 5 loops of grown E coli is taken on agar plates and added to the sterilized media of 150mL
in 1000mL shake flask. (You may need 2 of 1000mL shake flask to ensure enough inoculums
needed).
2. Sterility must be sustained during transfer.
3. The media is grown at 350 rpm for 4 hours and it is assumed as exponential growth of E
coli.
4. At this stage, the seed cultures are assumed to be at its most active condition.
5. OD reading is taken for seed culture using spectrophotometer during this time.

7
b) Main experiment

1. 10% of inoculum to the main experiment media is transferred using aseptic technique. (For

instance, if the working volume is 150ml, therefore, 10% of inoculum would be15mL of seed
culture needed).
2. The shake flask is then capped (cotton plugged) and swabbed with 70% ethanol before
incubation in a thermostated rotary shaker at required rotational speed and temperature for 24
hours.

(iii) Sampling

1. Required amount of sample is transferred into the sampling tube with interval time for
every hour or every 2 or 3 hours.
2. 5 mL of sample is withdrawn every time sampling is done during fermentation for
measuring optical density (OD), glucose analysis and total cell number (biomass
concentration: g/L).
3. Table below is referred for planned usage of sample volume:

No. Sample Volume (μL) Use for

Name
1 OD 1000 Optical measurement using spectrophotometer
2 CDW 1000 For Cell Dry Weight measurement

(iv) Absorbance  Analysis (Optical Density) (OD)

1. 1 mL of sample is transferred into a cuvette and the optical density measurement ismade
using a spectrophotometer with the wavelength set at 600nm.
2. The spectrophotometer is calibrated to zero by blank consisting 1 mL chosen media.
3. This method is used to measure cell growth; higher number of cells means more
absorbance, which is caused by low transmittance and vice versa.

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(v) Cell  Dry Weight. (Biomass Concentration) (X) (g/L)

1. Dried centrifuge tubes is weighted and noted as initial mass. (Empty container)

2. 1.5 mL sample is added to weighted centrifuge tube.
3. The sample is centrifuged at 10,000 rpm and at T of 4°C. For 20 minutes.
4. The supernatant is taken out and washing with distilled water and centrifuging may be
repeated.
5. The centrifuge tube is dried (left with biomass only) in oven at 80°C for overnight.
6. The dried centrifuged tubes is left in desiccators for half an hour.
7. The centrifuge tube weighted and note this as final mass (with biomass = Cell Dry
Weight).
Cell Dry Weight = Final mass –Initial mass

9
RESULTS

NO TIME (h) CDW ABSORBANCE

Empty Dried m2- m1 OD (10 Real OD
Centrifuge Centrifuge times
tube, (m1) tube + dilution)
sample, (m2)
1 0 1.0867 1.0906 0.0039 -0.159 0.056
2 1 1.0628 1.0670 0.0042 -0.079 1.504
3 2 1.0704 1.0762 0.0058 0.114 1.572
4 3 1.0660 1.0741 0.0081 0.426 1.815
5 4 1.0658 1.0740 0.0082 0.677 2.236
6 6 1.0712 1.0769 0.0057 0.679 2.303
7 8 1.0706 1.0770 0.0064 0.769 2.370
8 10 1.0614 1.0704 0.0090 0.801 2.471
9 12 1.0708 1.0783 0.0075 0.892 2.513
10 14 1.0823 1.0911 0.0088 1.034 2.610
11 16 1.0679 1.0763 0.0084 1.197 2.621
12 18 1.0682 1.0770 0.0088 1.298 2.707
13 20 1.0705 1.0790 0.0085 1.423 2.650
14 22 1.0759 1.0855 0.0096 1.437 2.670
15 24 1.0753 1.0853 0.010 1.624 2.645

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 Graph
Graph
OD Real
(10OD
times
vsdilution)
Time
23 vs Time (h)

2.5
1.5
OD (10 times dilution)

2
1
1.5
OD

0.5 1

0.5
0
0 5 10 15 20 25 30
0
0 5 10 15 20 25 30
-0.5
Time (h)
Time (h)

Figure 1: Graph of growth curve (for diluted sample)

Figure 2: Graph of growth curve (for no diluted sample)

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Graph Cell Dry Weight against Time

Dried (m2 - m1) X(g/L) Ln X

Empty ln X vs Time
Centrifuge
Time (h) Centrifuge
2 tube +
tube, (m1)
1.8 f(x) = 0.03 x + 1.28 sample, (m2)
0 1.6 1.0867 1.0906 0.0039 2.600 0.9555
1.4
1 1.2 1.0628 1.0670 0.0042 2.800 1.0296
1
ln X

2 1.0704 1.0762 0.0058 3.867 1.3524

0.8

3
0.6 1.0660 1.0741 0.0081 5.400 1.6864
0.4
4 0.2 1.0658 1.0740 0.0082 5.467 1.6987
0
6 0 1.0712
5 10 1.0769 15 0.0057
20 3.800
25 30 1.3350
Time (h)
8 1.0706 1.0770 0.0064 4.267 1.4508

10 1.0614 1.0704 0.0090 6.000 1.7918

12 1.0708 1.0783 0.0075 5.000 1.6094

14 1.0823 1.0911
Figure 3: Graph 0.0088
ln X versus time 5.867 1.7693

16 1.0679 1.0763 0.0084 5.600 1.7228

18 1.0682 1.0770 0.0088 5.867 1.7693

20 1.0705 1.0790 0.0085 5.667 1.7346

22 1.0759 1.0855 0.0096 6.400 1.8563

24 1.0753 1.0853 0.010 6.667 1.8971

∆y
Specific growth, µnet = gradient = = 0.028
∆x
h-1

Graph of Exponential Growth Phase

Time (h) Ln X Ln X/X0
0 0.9555 0.3675
1 1.0296 0.3960
Graph ln X/X0 vs time (h)
2 1.3524 0.5202
3 0.7 1.6864 0.6486
f(x) = 0.08 x + 0.35
4 0.6 1.6987 0.6533
0.5

0.4
ln X/X0

0.3

0.2

0.1
12
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Time (h)
Maximum growth rate, µmax = slope of the graph

µmax = 0.0824h-1

SAMPLE CALCULATION

1. Sample calculation of Cell Dry Weight, X (g/L):

[ ( Mass of centrifugetube+ sample )−( Mass of empty centrifugetube ) ]
=
0.0015 L
(m2−m1 )
=
0.0015 L
(1.0906−1.0867)
=
0,0015
= 0.0039

2. To determine the Specific growth rate, based on Figure 3 by using the linear equation
obtained from the graph of ln x value against time (y = 0.028x + 1.2783).
Specific growth, µnet = gradient = 0.028 h-1

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 3. To determine the maximum growth rate, based on Figure 4 by using the linear
equation obtained from the graph of ln X/Xo value against time (y = 0.0824x + 0.3523).
Maximum growth rate, µmax = slope of the graph = 0.0824 h-1
ln 2
4. Doubling time, td= µ
net

ln 2
=
0.028
= 24.76 h

DISCUSSION

In this experiment, E coli was selected as the cell and it is being cultivated in shake
flask. The flask is shaken during cultivation ensuring it is homogenous. The flask is shaken
continuously within 24 hour in order to make the microorganisms growth. Microorganisms
need more time to growth. Glycerol and glucose was being supplied as carbon sources and
both of it acts as a substrate nutrients for the cells.
In order to analyse the concentration of the cell inside the flask, absorbance reading
for the optical density is taken from the spectrophotometer. The higher the absorbance
reading shows that the higher number of cell presence inside the flask at a particular time. As
for this experiment, the absorbance reading is increase from the beginning of the experiment.

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It can be proven from figure 1 and figure 2. Figure 1 show the growth curve for the diluted
sample while figure 2 show the growth curve for no diluted sample. From figure 2, the
absorbance value decrease slightly at 20th hour. It can be explained that the number of cell
increase throughout the cultivation indicating that the cell is growing. In the other hand, the
decrease in cell number indicating that the cell growth has reach its deceleration phase where
the growth of the cell is started to slow down. The decelerating growth phase is where the
culture is in a transient state. During this stage there are feed/back mechanisms that regulate
the bacterial enzymes involved in key metabolic steps to enable the bacteria to withstand
starvation. There is much turnover of protein for the culture to cope with this period of low
substrate availability. In cell growth, the cell will go through several phases like lag,
exponential, deceleration, stationary and death phase.
Another analysis that can be performed in this experiment is to analyse the cell
sample is by taking the dry weight of the cell. In this method, the cell is being taken out from
cultivation flask and transferred into centrifuge tube. The tube is the being centrifuged to
separate the supernatant with the cell. The remained cell is then being dried inside an oven
for 24 hours. The dry cell weight is finally taken to know the weight of the cell that present at
particular time during the cultivation. In this experiment, the cell dry weight is increased from
0th hour until 4th hour and gradually decreased from the 9th hour to 8th hour. For The cell dry
weight should increase when the number of cell increased inside the shake flask.

CONCLUSION

This experiment was carried out to investigate the growth kinetics of

microorganism in shake flask. Microorganisms will go through several phases during their
growth such as lag phase, exponential or log phase, stationary phase, and death phase.
Analysts has been made to obtain the kinetics of the cell and duration for each phase. Then,
a growth curve including lag, log, stationary and death phases were plotted. The others
parameters that being studied in this experiment includes cell concentration, absorbance
reading, and cell dry weight (CDW). From the graph plotted and equations that have been
given, the Monod parameters such as µnet, µmax and td were calculated even though there is

15
some parameters that cannot be calculated because of technical problem that we cannot
avoid. This experiment succeed.

RECOMMENDATION

1. Avoid contamination when handling the biomass by practicing excellent aseptic

techniques.
2. This experiment must be carried out under the laminar flow to prevent any
contamination to the culture.
3. Cuvette must be in clean condition to obtain most accurate spectrophotometer reading
during protein test
4. The supernatant of cell concentration should be taken out carefully without any taking
out of the biomass.
5. Always sterilize the mouth of shake flask and cotton plugged with flame from Bunsen
burner to avoid any contamination.
6. Make a dilution if the reading of spectrophotometer is more than 1 and take the
Optical Density reading again.

REFERENCES

1) Jha. N, Growth of microorganisms. Retrieved from

http://www.biologydiscussion.com/microorganisms/growth-of-microorganisms-with-
diagram/10138 on 7 November 2016.

2) The different factors influencing the growth of microorganisms. Retrieved from

https://www.academia.edu/8830229/THE_DIFFERENT_FACTORS_INFLUENCING_THE
_GROWTH_OF_MICROORGANISMS_AND_THE_EFFECT_OF_TEMPERATURE_ON_

16
THE_GROWTH_AND_SURVIVAL_OF_MICROORGANISMS_IN_FOOD on 10
November 2016.

3) Growth requirements for microorganisms. Retrieved from

https://www.cliffsnotes.com/study-guides/biology/microbiology/microbial-cultivation-and-
growth/growth-requirements-for-microorganisms on 10 November 2016.

4) Fogler, Scott H. Elements of Chemical Reaction Engineering, 4 th ed. Englewood Cliffs,

NJ: Prentice hall, 2011.

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