CHE506

Edited May 2020

Lab #6: Growth Kinetics Study of Microorganism in Shake Flask

Objectives:

• To study/observe the growth kinetics of microorganism in shake flask experiment

• To construct a growth curve including lag, log, stationary and death phases.

1. Introduction

In the shake flask fermentation, the culture flasks (usually Erlenmeyer) of 250 or 500 mL or larger
are used for growing microorganisms. Shake flask fermentation is the cheapest and simplest technique
to grow bacteria or fungi, aerobically, in small volumes of nutrient broth. The broth is poured into
Erlenmeyer Flasks equipped with cotton-wool stoppers, and autoclaved. After cooling, some microbes
are "seeded" into the flask, and it is placed on a rotary shaker. The shaking agitates the content and so
ensures aeration, so that the microbes could breathe. These flasks are shaken, generally, by an
incubator shaker at a suitable agitation speed, which is usually in rotation per minute (rpm). Shaken
cultures are usually applied to aerobic processes. In general, filamentous microorganisms are grown to
produce secondary metabolites, which begins 1 to 3 days after inoculation and continues 3 to 4 days
thereafter, for instance. In all such cases, the shaken cultures are used for strain improvement as well
as for determination of the optimum conditions for the fermentation process. In many industrial
processes, it is also used for the initial stages of inoculum development. Shaken cultures are a
convenient method of growing microorganisms in submerged cultures under aerobic conditions created
by shaking; it is a small-scale equivalent of stirred tank bioreactor. Both the devices are extensively
used with filamentous microorganisms and, often, with other types of microorganisms as well.

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.

In a batch culture, there is neither input supplied, nor output generated throughout the
fermentation. The medium culture is initially inoculated with the microorganism. The growth keeps
increasing until at certain extent, the growth is inhibited because of the decreasing substrate
concentration and the presence of toxic metabolites.

Figure 1 shows the phases of a typical growth curve of E.coli in a batch culture. 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.

The second phase is exponential 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, Td since the growth at this time is the most constant and
ideal.

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Figure 1: Phases of a typical growth curve of E.coli in a batch culture.

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. Finally, the growth declines from its stationary
phase due to the cells lysation. This is indicated by the decrease of the viable cell number.

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 (Optical Density at wavelength of 600 nm) is 5 to 8 under normal
shaking incubation conditions. TB is commonly used for protein expression and plasmid production in
a laboratory scale.

2. Theories

Rate of microbial growth µnet is characterized by a specific growth rate:

1 𝑑𝑑𝑑𝑑
𝜇𝜇𝑛𝑛𝑛𝑛𝑛𝑛 = [ℎ𝑟𝑟 −1 ]
𝑋𝑋 𝑑𝑑𝑑𝑑

Mass doubling time (𝜏𝜏𝑑𝑑 ) is calculated based on cell numbers and the net specific rate of replication

ln 2
𝜏𝜏𝑑𝑑 = [ℎ𝑟𝑟]
𝜇𝜇𝑛𝑛𝑛𝑛𝑛𝑛

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3. Apparatus and Reagent

a) Microbe: Escherichia coli

b) Shake flask (250 mL flasks and 500 mL flasks)
c) Eppendorf tubes/falcon tube (1.5 mL)
d) Cuvettes (spectrophotometer)
e) Thermostated rotary shaker/incubator shaker
f) Refrigerated centrifuge
g) Media (for specific microbe)
h) Ethanol (70% ethanol for swabbing for sterility)
i) Spectrophotometer
j) Bunsen burner for sterility
k) Graduated flask for measuring media (1000 mL, 100 mL, 10 mL)
l) Laminar flow hood for sterility
m) Cotton plugged
n) pH meter

4. Experimental Procedures

(i) Preparation of media

- Media must be prepared according to the needs of microorganism used.

- Microorganism used is Escherichia coli. There are many kinds of media for E.coli for
instance Luria Bertani broth or Terrific Broth. Terrific Broth is a readied phosphate buffer
media.
- Prepare the Terrific Broth according to the recommended formula or recipe stated at the
chemical bottle and listed in Table 1.
- The example readied recipe for the broths are as the following. Please further read the
instruction on the bottle.

Table 1: Broth

Name of Broth Brand Recipe (g/L

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

- Terrific Broth (TB) preparation and sterilization of media

• Follow the TB recipe as stated at the bottle.
• Pour 20 mL of prepared media in 100 mL of an Erlenmeyer flask and 150 mL of
prepared media in 500 mL of an Erlenmeyer flask. Now you have smaller flask for
inoculum preparation and bigger flask for the main experiment (growth study of
E.coli). Cap the flask with cotton plugs and followed by aluminium foil.
• Tag all the flasks and micropipette tip’s boxes with indicator tape. Autoclave the
media (both flask) and the micropipettes tips in an autoclave at 121°C for 20 minutes.
The whole process may take between 3 to 4 hours due to heating ramping rate,
holding time and cooling down the autoclave to room temperature after completed the
sterilization process.
• Glycerol and media can be autoclaved together.
• pH reading should be near 7 as the media is a readied phosphate buffer solution. You
may verify this.

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- Determine the optical density (OD) of the media.

After the media reach the room temperature, take 1 mL of media from the 100 mL
Erlenmeyer flask and dilute it with 9 mL of deionized water (DW). This will give a dilution
factor (DF) of 10. Then measure the OD using spectrophotometer at 600 nm
wavelength. The blank for the OD measurement is DW. The value of Real OD for this
measurement in noted as ODbm. (optical density for blank media). Then the media in
flask is ready for seed culture preparation.

(ii) Preparation of cell culture

Cell culture used must be maintained on an agar plate and liquid broth for the inoculum
preparation. A suitable media is needed in order to ensure that the microorganism is growing.
Inoculum preparation refers to seed culture which will be the feed for the main experiment.
Normally, the size of inoculum prepared is 10-20% of the working volume in bioreactor.

a) Seed culture preparation (inoculum)

Take 5 loops of grown E.coli on agar plates and added to the sterilized media of 150 mL in
1000 mL Erlenmeyer flask. You may need 2 of 1000 mL Erlenmeyer flask to ensure enough
inoculum. Sterility must be sustained during transfer. However, for this purpose of this
experimental work, the above steps have been conducted by the Lab Assistant. Hence, you
are supplied with 2 mL of E-coli in Eppendorf tubes.

The next step is to transfer the 2 mL (2 tubes) of E-coli into a 20 mL working volume of
100 mL shake flask. Sterility must be sustained during transfer. Watch the inoculum preparation
demonstration video:

- https://drive.google.com/file/d/12EtTc7GzcCf6pWtlqJ1Swe84hGtNI0yU/view?usp=sharing
- https://drive.google.com/open?id=1DjcAFpAySrhJQai_5JSdhaa2tP8gAxiM

Grow the bacteria at 300 rpm for 4 hours assuming exponential growth of E.coli. At this stage,
the seed cultures are assumed to be at its most active condition. Take note to measure the
optical density (OD) for the seed culture before (has been explain in step section 4(i)) and after
the fermentation using a spectrophotometer. The summary of the experimental parameters
for seed culture preparation is listed in Table 2.

Table 2: Experimental parameters for seed culture preparation

Temperature Shaking Shake Filling/ Media Inoculum pH Carbon

(°C) frequency flask working type percentage source
(rpm) size volume
(mL)
2 Eppendorf
37 300 100 20 TB 7 Glycerol
tubes

b) Main experiment (Growth study of E-Coli)

Using aseptic technique, transfer all the inoculum (between 10 – 20 % by volume) to the main
experiment media. For instance, if the working volume is 150 mL, therefore, 10 – 20 % of
inoculum would be 15 – 30 mL of seed culture is needed.

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Swirl the flask as to mii up the bacteria and the media. Next, take 1 mL of the sample for OD
and 1 mL for Cell Dry Weight measurement (CDW). This is considered at measurement at T=0.
Watch the demonstration at:

https://drive.google.com/file/d/10zeUEETqkH30rVrR85kAvxidojDI2-9V/view?usp=sharing

The shake flask is then capped (cotton plugged) and swabbed with 70% ethanol before
incubation in a thermostated rotary shaker at 300 rpm and temperature of 37°C for 24 hours.
Table 3 summarize the experimental parameters for the growth study of E.coli.

Table 3: Experimental parameters for the growth study of E.coli.

Temperature Shaking Shake Filling/ Media Inoculum pH Carbon

(°C) frequency flask working type percentage source
(rpm) size volume (%)
(mL)
37 300 500 150 TB 10-20 7 Glycerol
.

(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. A 1 mL of sample is withdrawn every time sampling is done during fermentation for

measuring OD and glucose analysis while another 1 mL sample is withdrawn for measuring
the total cell number (biomass concentration: g/L).

3. Refer to Table 4 below for planned usage of sample volume.

Table 4: Sampling amount and purpose

No Sample Name Volume (uL) Use for:

Optical density measurement using
1 OD 1000
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 is made
using a spectrophotometer with the wavelength set at 600 nm.

2. The spectrophotometer is calibrated to zero by blank consisting 1 mL chosen media.

However, to avoid the effect of deterioration of media with respect to time, the blank is
replaced with DW, and the absorbance of the blank media (ODbm) will be deducted from the
obtained absorbance for the sample. (subjected to 10 DF).

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.

Suggested method:

A tenth-time dilution (DF=10) is proposed for the OD measurement by using

spectrophotometer. For instance, with 1 mL sample, take only 100 uL sample being added to
900 uL of DW for OD measurement in 1000 uL cuvette. Or dilute the 1 mL sample with 9 mL
SW. (1 mL = 1000 µL).
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(v) Cell Dry Weight (CDW) (Biomass Concentration) (X) (g/L)

1. Weigh dried centrifuge tubes and note this as initial mass (empty container).

2. 1 mL sample is added to weighted centrifuge tube.

3. The sample is centrifuged at 10,000 rpm and at T=4°C for 20 minutes

4. Take out the supernatant and you may repeat washing with distilled water and centrifuging

5. Dry the centrifuge tube (left with biomass only) in oven at 80°C for overnight

6. Leave the dried centrifuged tubes in a desiccator.

7. Weigh the centrifuge tube and note this as final mass (with biomass = Cell Dry Weight)

CDW = Final mass – Initial mass

Alternative method

1. Aluminum weight of boat are dried in an oven at 80C for 6-8 hours and placed in a
desiccator containing a drying agent for cooling before weighing (for 30 min).

2. The cell pellet (after sample is centrifuged at 10,000 rpm) is suspended in 10 mL centrifuge
tube with distilled water.

3. The cell then transferred to aluminium foil boat. The tube was rinsed with water and placed
in an oven at 80°C for overnight.

4. The sample is then removed from the oven with tongs and placed in a desiccator to cool
and weighed rapidly on an analytical balance. The weight of the cell pellet is recorded.

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(vi) Proposed Table Data Collection Seed/Inoculum

Seed/Inoculum

No Time ODread (DF =10) Real OD =

ODread x 10
1 Initial (T=0 h) *
2 Final (T= 4 h)
* This value is referring to blank media = ODbm

Main Experiment (Growth study of E-coli)

No Time ODread Real OD OD (Abs) = Empty Dried CDW, X (g/L)

(h) (DF =10) = ODread Real OD - centrifuge centrifuge (m2-m1) x
x 10 ODbm tube, m1 tube, m2 1000
(g) (g)
1. 0
2. 0.5
3. 1
4. 1.5
5. 2
6. 3
7. 4
8. 6
9. 8
10. 10
11. 12
12. 14
13. 16
14. 18
15. 20
16. 22
17. 24

6. References

1. Shuler, Michael L., and Fikret Kargi (2002). Bioprocess Engineering. New York: Prentice
Hall.
2. Garvie, Ellen I. (1995). The growth of Escherichia coli in buffer substrate and distilled water.
Journal of Bacteriology, 69 (4),pg 393-398.

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