Lab Report E.coli

RESULT
Blank Media : 0.222 nm
Diluted Blank Media : 0.022 x 10 : 0.22 nm
Table 1 : Seed Culture / Inoculum
Time (hour) Real Optical Density (nm)

0 0.158
4 1.800
Table 2 : Absorbance Optical Density Reading for 9 Hours
No Time (hour) Absorbance Optical Density, Actual Absorbance Optical Density,

OD Actual OD (nm)
1 0 0.86 0.64
2 1 2.00 1.78
3 2 3.16 2.94
4 3 3.63 3.41
5 4 4.33 4.11
6 6 3.63 5.07
7 7 5.26 5.04
8 8 5.55 5.33
9 9 5.59 5.37
10 10 5.70 5.48
Table 3 : Cell Dry Weight (Concentration of the Biomass)
No Time Empty Dried Cell Dry Cell Mass Ln X Ln (X/Xo)

(hour) Centrifuge Centrifuge Weight Concentration,
Tube, m1, Tube + (m2 – m1), X, (g/L)
(g) Sample, m2 (g)
(g)
1 0 1.0698 1.0706 0.0008 0.8 -0.2231 0
2 1 1.0737 1.0755 0.0018 1.8 0.5878 0.8109
3 2 1.0695 1.0719 0.0024 2.4 0.8755 1.0986
4 3 1.0661 1.0693 0.0032 3.2 1.1632 1.3863
5 4 1.0826 1.0845 0.0019 1.9 0.6419 0.8650
6 5 1.0712 1.0731 0.0019 1.9 0.6419 0.8650
7 6 1.0740 1.0766 0.0026 2.6 0.9555 1.1787
8 7 1.0727 1.0748 0.0021 2.1 0.7419 0.9651
9 8 1.0695 1.0716 0.0021 2.1 0.7419 0.9651
10 9 1.0761 1.0787 0.0026 2.54 0.9555 1.1787
Table 4 : Net Growth Rate at Different Time
No Time (hour) Cell Mass Concentration, X, Ln X μnet ( h−1 )

(g/l)
1 0 0.8 -0.2231 0.0000
2 1 1.8 0.5878 0.8109
3 2 2.4 0.8755 0.2877
4 3 3.2 1.1632 0.2877
5 4 1.9 0.6419 -0.5213
6 5 1.9 0.6419 0.0000
7 6 2.6 0.9555 0.3136
8 7 2.1 0.7419 -0.2136
9 8 2.1 0.7419 0.0000
10 9 2.6 0.9555 0.2136
Actual Absorbance Optical Density (nm)
6
0
0 1 2 3 4 5 6 7 8 9
Time (h)
Figure 1 : Actual Absorbance Optical Density vs Time
3.5
Cell Mass Concentration, X (g/L)
2.5
1.5
0.5
0
0 1 2 3 4 5 6 7 8 9
Time (h)
Figure 2 : Cell Mass Concentration vs Time

6
Ln X/Xo
3
f(x) = 0.3 x − 0.28
2
0
0 1 2 3 4 5 6 7 8 9
Time (h)
Figure 3 : Ln X/Xo vs Time

CALCULATIONS
1. Sample Calculation for Determination Dry Cell Weight (g)

Cell Dry Weight ¿ Final Mass, m2 −¿ Initial Mass, m1
For 0th hour
Cell Dry Weight ¿ 1.0706 g – 1.0698 g
¿ 0.0008 g
2. Sample Calculation for Cell Mass Concentration, X (g/L)

Cell Dry Weight (g)
Cell Mass Concentration (g/L) ¿
Volume of Sample (L)
For 0th hour :
0.0008(g)
Cell Concentration (g/L) ¿
0.001(L)
¿ 0.8 g/L
3. Sample Calculation for Ln (X/Xo)
For 0th hour :
0.8 g / L
Ln (X/Xo) ¿ Ln
0.8 g / L
¿0
4. Sample Calculation for Maximum Growth Rate, Mmax

Based on the graph of Ln (X/Xo) vs Time, the slope of the graph is a μmax value.
Therefore the μmax is 0.2976 hr −1
5. Sample Calculation for Doubling Time, td

Doubling time is the time required to double the microbial mass.
ln (2)
td ¿
μmax
td ¿ 2.3291 hr
6. Sample Calculation for New Growth Rate, μnet
(ln X 2 – ln X 1)
μnet ¿
t 2−t
1
0.5878 – (−0.2231 )
μnet ¿
1−0
¿ 0.8109 h−1
DISCUSSION
The objective of this experiment is to observe the growth kinetics of microorganism in

the shake flask. This experiment is also conducted to construct and discuss the growth curve of
the E.coli that is chosen as the cultures which is including different phases of the growth. E.coli
is being selected in this experiment as it is easy to culture due to its characteristics which is it is
able to grow anaerobically and aerobically. However, in this experiment the E.coli grows in
aerobic condition. In this part, E.coli is cultivated in a flask that is being shaken in an incubator
shaker in order to enhance the seeding of the cell. Besides, this experiment is using the batch
culture process which is there is neither input ore output generated throughout the process.
During cultivation that used fermentation process, the cell sample is taken to examine
concentration of the cell and the cell dry weight.
The absorbance reading for the optical density is recorded using the spectrometer to
determine the concentration of the cell inside the flask. The optical reading is shows the growth
of the E.coli. From the curve growth plotted in Figure 1, the reading of the absorbance increase
exponentially from the beginning which is from the 0 hour to 2 hours and continues to rise from
2 hours to 5 hours. During this stage, the number of cell increase due to the cell of the E.coli is
managed to growth all through the cultivation.
Moreover, this ascending of the reading of the absorbance indicates that the E.coli is gone
through the exponential phase where it grows rapidly due to existences of the nutrient provided
and from the phase, the maximum growth rate (μmax) can be determined from the slope of Figure
3 which yield μmax is 0.2976 hr −1 and from the value of μmax , the mass doubling time (td) may be
obtained from the Equation 1 which gave the value 2.3291 hours. However, the curve growth
supposedly started with the lag phase which indicates that the cell is adapt to the new
environment or cultural conditions that means there is lack of growth or slow growth. This
situation is maybe occurred due to the higher possibility of the cell is have been adapted the
condition only for a short time which means it happened before the first hour of absorbance
reading. Thus, the lag phase cannot be detected in this experiment.
Next, from 5th hours to 6th hours, the readings are slightly reduced which are 5.07 and 5.04
respectively. This phase is called the deceleration phase where it happen only for a short time.
During this phase, the growth of E.coli decrease a little bit due to the descending of the main
nutrients and the production of the toxic by-product begin to produce in the flask.
Furthermore, based on the Figure 1, it shows that the stationary phase start to occur at
time 7 to time 9 where the OD is slightly constant and have a little bit difference which is 5.33 to
5.48. In this phase, the growth is concluded to be zero since there is no cell division. Even
though there is no net increase in number of cells, the cells is still active but it only less
metabolically active due to the increasing in competition for nutrients. It can be seen from the
Figure 1, the optical density is increase a little bit. The death phase should happen after the
stationary phase. However, the experiments have been stopped before that phase occurs due to
lack of time. In this phase, the number of living cells decreases exponentially and population
growth experiences a sharp decline. It is also create the net loss viable cells where the cells will
be destroyed by lysis. (https://www.facebook.com/thoughtcodotcom, 2018)
Above and beyond, the value of μnet can be obtained from the Figure 3 for the different
phase which produce the value of μnet, exponential phase = μmax = 0.2976 h-1, μnet, descending
phase = 0.3136 h-1, μnet, stationary phase = 0.0000 h-1. Based on the theory, the value of
exponential phase must be the highest value compare to other phase. (Growth Kinetics of
Microorganisms in a Shake Flask,2018). However, on the contrary based on the value obtained,
the descending phase shows the higher value compared to the exponential phase. This type of
error may be generated from the improper technique while conducting the experiment which
comes from the human error in taking the data for the cell dry weight. This situation may be also
happen because of the incorrect way of handling the aseptic technique when do biomass
concentration result on the contamination of the E.coli. However, this experiment has been
conducted successfully as the most of the objectives have been achieved.
REFERENCES
https://www.facebook.com/thoughtcodotcom. (2018). The Bacterial Growth Curve and the
Factors Affecting Microbial Growth. Retrieved from ThoughtCo website:
https://www.thoughtco.com/bacterial-growth-curve-phases-4172692
‌Bacterial Growth Curve Analysis and its Environmental Applications | Protocol. (2019).
Retrieved from Jove.com website: https://www.jove.com/science-
education/10100/bacterial-growth-curve-analysis-and-its-environmental-applications
‌Growth Kinetics of Microorganisms in a Shake Flask. (2018). Retrieved November 16, 2019,
from UKEssays.com website: https://www.ukessays.com/essays/biology/growth-kinetics-
study-of-microorganism-in-shake-flask-biology-essay.php
E. coli – the biotech bacterium. (2014). Retrieved from Science Learning Hub website:
https://www.sciencelearn.org.nz/resources/1899-e-coli-the-biotech-bacterium
Microbial Growth. (2010). Retrieved November 16, 2019, from Montana.edu website:
https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002
/section002/black/page001.html

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RESULT

Blank Media : 0.222 nm

Diluted Blank Media : 0.022 x 10 : 0.22 nm

Table 1 : Seed Culture / Inoculum

Time (hour) Real Optical Density (nm)

Table 2 : Absorbance Optical Density Reading for 9 Hours

No Time (hour) Absorbance Optical Density, Actual Absorbance Optical Density,

Table 3 : Cell Dry Weight (Concentration of the Biomass)

No Time Empty Dried Cell Dry Cell Mass Ln X Ln (X/Xo)

Table 4 : Net Growth Rate at Different Time

No Time (hour) Cell Mass Concentration, X, Ln X μnet ( h−1 )

Figure 1 : Actual Absorbance Optical Density vs Time

Figure 2 : Cell Mass Concentration vs Time

Figure 3 : Ln X/Xo vs Time

1. Sample Calculation for Determination Dry Cell Weight (g)

2. Sample Calculation for Cell Mass Concentration, X (g/L)

4. Sample Calculation for Maximum Growth Rate, Mmax

5. Sample Calculation for Doubling Time, td

The objective of this experiment is to observe the growth kinetics of microorganism in

https://www.facebook.com/thoughtcodotcom. (2018). The Bacterial Growth Curve and the

Factors Affecting Microbial Growth. Retrieved from ThoughtCo website:

Retrieved from Jove.com website: https://www.jove.com/science-

from UKEssays.com website: https://www.ukessays.com/essays/biology/growth-kinetics-

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