CBE 661 Lab 1

Edited 2 Feb 2015

Lab Manual

Laboratory 1: 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.

To determine the Monod parameters of maximum growth rate (max),

yield of substrate (Yx/s), mass doubling time (td), saturation constant
(Ks), specific growth rate (net).

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 Shaker machine.
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 r.p.m. Shaken cultures are usually applied to aerobic processes. In general,
filamentous microorganisms are grown for the production of 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.

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

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.
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, and doubling time, d since the growth at this time is the
most constant and ideal.
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 5 to 8 under normal
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Lab Manual

shaking incubation conditions. TB is commonly used for protein expression and plasmid
production in a laboratory scale. (TB broth)

2. Theories
Rate of microbial growth net is characterized by specific growth rate:

net

1 dX
X dt

[1/h]

Yield Coefficients ( Y X / s ) are defined based on the amount of consumption of another material

YX / s =

X
S

[g cells/g substrate]

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

d =

ln 2

[h]

net

For substrate limited growth Monod equation is applicable in cellular system. Monod equation is
as the following:

g =

m S

[1/h]

Ks + S

m = maximum specific growth rate when S >> K s

g = net when endogeneous metabolism is unimportant
K s = saturation constant or half-velocity constant
K s = S when g = m
S>> K s , g = m
S<< K s , g =

m S
Ks + S

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3. Apparatus and Reagent

Microbe: Escherichia coli

Shake flask (250mL flasks and 1000 mL flasks)
Eppendorf tubes/falcon tube (1.5mL)
Cuvettes (spectrophotometer)
Thermostated rotary shaker/Incubator shaker
Refrigerated Centrifuge
Media (for specific microbe)
Ethanol (70% ethanol for swabbing for sterility)
Spectrophotometer
Bunsen Burner for sterility
Graduated Flask for measuring media (1000mL, 100mL, 10mL)
Laminar Flow hood for sterility
Biochemical Analyzer
HPLC for product measurement like ethanol
Cotton plugged
pH meter

4. Experimental Procedures
The experimental parameters will be as the following

Group

Shaking Shake Filling/Working

Temperature
Media Inoculum
frequency flask
Volume
o
( C)
Type percentage
(rpm)
size
(mL)

pH

TB
1

37

350

500

150

Glycerol
10

LB
2

37

350

500

150

TB

Glucose
Glycerol
10

LB
3

37

350

500

150

37

350

500

150

37

350

500

150

37

350

500

150

TB
LB
TB
LB
TB
LB
TB
LB

Carbon
source

Glucose
10

10

10

10

Glycerol
Glucose
Glycerol
Glucose
Glycerol
Glucose
Glycerol
Glucose

Table 1: Experimental Parameters

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(i)

Lab Manual

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 chosen broth according to the recommended formula or recipe stated at
the chemical bottle.
The example readied recipe for the broths are as the following:
Please further read the instruction of bottle
No
1
2

Name of Broth
Terrific Broth
Luria
(Lennox)

Bertani

Brand
SRL Chem
or Merck
SRL Chem

Recipe (g/L
47.60 g to be filled up to 1L volume
Glycerol as carbon source (4mL/1L)
20 .0 g to be filled up to 1L volume
Glucose as carbon source (10g/L)

Table 2: Broth
a) Terrific Broth preparation
Follow the recipe as stated at the bottle.
Autoclave the media at 121 oC for 20 minutes
Glycerol and media can be autoclaved together.
pH reading should be near 7 as the media is a readied phosphate buffer
solution
b) Luria Bertani (Lennox) preparation
Follow the recipe as stated at the bottle for LB media.
A certain amount of phosphate buffer is added to give a specific pH which is
pH 7. Refer to table below.
Prepare glucose solution and it should be autoclaved separately and cooled
down in a different bottle.
Autoclave distilled water which will be used for filling up volume to the needed
volume prepared.
When both solutions are cooled, then only both solutions are added.

No
1
2

Phosphate buffer
components
K2HPO4
KH2PO4

Concentration
8 g/L
3 g/L

Table 3: Buffer recipe according to 50 mM Buffer Strength (Buffer

calculator)

(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
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microorganism is growing. Inoculum preparation refers to seed culture which will be

the feed for the main experiment.
a) Seed culture preparation (inoculum)
Take 5 loops of grown E coli 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 inoculum needed)
Sterility must be sustained during transfer.
Grow the media at 350 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 the OD for seed culture using spectrophotometer
Shake
flask
Filling/
Shaking
Temperature
size
Working
frequency
(oC)
Volume
(rpm)
(mL)

37

350

1000

150

pH
Media Inoculum
Type percentage

TB

5 loops
from agar
plate

Fermentation Initial
time
and final
Carbon
(h)
OD of
source
seed
culture
**
Glycerol

OD
initial

OD final

Table 4
Take note: (please prepare enough seeds for all main experiments)
** Please take note the initial OD (after 5 loops inoculation) and final OD
(after 4 hours of fermentation)

b) Main experiment
Using aseptic technique, transfer 10% of inoculum to the main experiment media.
For instance, if the working volume is 150ml, therefore, 10% of inoculum would be
15mL of seed culture needed
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.
The main experiment is stated in Table 1.

(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. Refer to Table below for planned usage of sample volume:
No
1
2

Sample
Name
OD
CDW

Volume
(uL)
1000
1000

Use for
Optical measurement using spectrophotometer
For Cell Dry Weight measurement
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4

Lab Manual

S
P

1500
remaining

Glucose Analysis
Product analysis i.e ethanol

Table 5: Sampling
v)

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 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.
Suggested method:
Certain tenth-time dilution is proposed for the OD measurement by using
spectrophotometer. For instance, with 1 mL sample, take only 100 uL sampl e being
added to 900 uL of Distilled Water for OD measurement in 1000 uL Cuvette.

vi)

Cell Dry Weight. (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 of 4 oC. 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 oC for overnight
6. Leave the dried centrifuged tubes in dessicator.
7. Weigh the centrifuge tube and note this as final mass (with biomass = Cell Dry
Weight)
Cell Dry Weight = Final mass Initial mass

Alternative method
1. Aluminum weight of boat are dried in an oven at 80C for 6-8 hours and placed in
a dessicator containing a drying agent for cooling before weighing (for
30min).
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 aluminum foil boat. The tube was rinsed with water
and placed in an oven at 80C for overnight.
4. The sample is then removed from the oven with tongs and placed in a dessicator
to cool and weighed rapidly on an analytical balance. The weight of the cell pellet
is recorded.

vii)

Glucose Analysis. (Substrate Concentration) (S) (g/L)

1. Sample of 1.5 mL is transferred into the micro centrifuge tube and refrigerated
centrifuged for 10 minutes at 10,000 rpm.
2. Then, the supernatant is taken out into cuvette and put onto turntable of YSI 2700
Select Biochemical Analyzer for direct analysis of glucose (dextrose)
concentration.
3. The glucose analysis is based on Glucose Oxidase that has been immobilized in
the YSI Dextrose Membrane (YSI 2365).

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Suggested method:
This method is to analyze the concentration of S: substrate which is glucose as the
main source of carbon. The glucose being consumed is monitored and denoted as S
in the unit of g/L.
Take note: only for media having glucose only.
viii)

Product analysis (optional)

1. The remaining sample is transferred into the centrifuge tube and centrifuged for 10
minutes at 10,000 rpm.
2. Then, the supernatant is taken out to analyze the product desired by following the
suitable methods.

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ix)

Lab Manual

Proposed Table Data Collection

Seed/Inoculum
No

Time
(h)

OD (10
times
dilution)

Real OD

Main experiment

Absorbance
Optical
Density
No

Time
(h)

OD
(10 times
dilution)
OD read

1
2
3
4
5
6
7
8
9

Absorbance
Empty
Centrifuge
Real OD

m1

Dried Centrifuge
tube + sample
m2

Cell Dry Weight

X
(g/L)
(m2-m1)

Substrate
S
(g/L)

Product
P
(g/L)

(ODread
times 10)

0
3
6
9
12
15
18
21
24

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Lab Manual

5. Report: (100M)
Plagiarism is highly prohibited.

1
2
3
4
5
6
7
8
9
10
11
12

Abstract/Summary
Introduction
Aims/Objective
Theory
Apparatus
Methodology/Procedure
Results
Calculations
Discussion
Conclusion
Recommendation
Reference/Appendix

(5M)
(5M)
(5M)
(10M)
(5M)
(10M)
(10M)
(10M)
(20M)
(10M)
(5M)
(5M)

6. References
Buffer calculator: http://home.fuse.net/clymer/buffers/phos2.html
Shuler, Michael L., and Fikret Kargi. Bioprocess engineering. New York: Prentice Hall,
2002.
Garvie, Ellen I. "The growth of Escherichia coli in buffer substrate and distilled
water." Journal of bacteriology 69.4 (1955): 393.

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