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(CBE 661)










No. Title Allocated Marks Marks

1. Abstract/Summary 5
2. Introduction 5
3. Aims 5
4. Theory 5
5. Apparatus 5
6 Methodology/Procedure 10
7. Results 10
8. Calculations 10
9. Discussion 20
10. Conclusion 10
11. Recommendations 5
12. Reference 5
13. Appendices 5

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Date: Date:

Table of Contents


1 Abstract 3
2 Introduction 4
3 Objectives 5
4 Theory 5
5 Apparatus 8
6 Methodology/Procedure 8
7 Results 11
8 Calculations 17
9 Discussion 17
10 Conclusion 20
11 Recommendations 21
12 Reference 21
13 Appendices 22


In this experiment the study is for the growth kinetics of microorganisms in shake flask. E.coli is
grown in a LB and TB broth mediums and being fermented for 24 hours at 350 rpm and 37 0C.
Throughout the fermentation, the cell culture is taken out every 1 hours from 0 hour to 4th hour
and continued with every 2 hour until 20th hour and the absorbance analysis, glucose analysis
and cell dry weight are being performed. As for the optical density analysis, the absorbance
reading from the spectrophotometer is taken while for the glucose test, the reading of glucose
level is taken from the YSI 2700 Select Biochemical Analyzer. These substrate concentration
values will then being plotted on graph with growth rate. 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. For the optical density of the cell, the absorbance value showed an increment which
indicating that the cell was growing and number of cell is increased in the shake flask. The growth
rate, however, cannot be determined as the absorbance values were increased and decreased
unevenly and calculation of rate of microbial growth cannot be made as the data for cell dry weight
concentration, X are not consistent giving some of the results negative value thus some of the
rate of growth, μ and substrate constant, Ks cannot be calculated. Supposedly, as the number of
cell increased inside the shake flask, the cell dry weight also should be increased. Therefore no
conclusion can be made about the cell growth rate in the shake flask. Another reason because
the electronic weight balance shows different reading when weighted twice.


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.

To ensure the prevention of any contamination to the culture, shake flask must be plugged.
Different plug can be made of cotton-wool, glass wool, polyurethane foam, gauze or synthetic
fibrous material. In this experiment, we used cotton plugged. The plug function to prevent airborne
microorganism from getting into the medium while at the same time allowing free flow of air enter
the flask.

The cultures are incubated at certain temperature 37o C and shaking frequency 350 rpm for 4
hours in an incubator shaker to achieve a required growth rate. The shaking agitates the medium
and the culture to keep the mixture relatively homogeneous and also to ensure aeration, creating
an aerobic condition. In batch culture, it is closed environment that means 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

The microorganism that we used to study in this experiment is E.coli. There are many specific
media for growth of E.coli but in this experiment we used Luria Bertani (LB) a Terrific Broth (TB)
media because it is the most commonly used medium in molecular biology for E. coli cell culture.
The relationship between the specific growth rate (μ) of a microbial population and the substrate
concentration (s), is an indispensable tool in all fields of microbiology, be it physiology, genetics,
ecology, or biotechnology, and therefore it is an important part of the basic teaching of
microbiology (Kovárová-Kovar. K, et. al, 1998)


– To study 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


Shake flask fermentation is one of the examples of batch fermentation. Batch culture is an
example of a closed culture system which contains an initial, limited amount of nutrient. The
inoculated culture will pass through a number of phases. After an inoculation there is a period
during which no growth appears to take place. This period is referred as the lag phase and may
be considered as a time of adaptation. In a commercial process, the length of the lag phase should
be reduced as much as possible. Following a period during which the cell gradually increases,
the cell grows at constant, maximum rate and this period is known as the exponential phase. The
exponential phase may be described by the equation below:

= µx ----------------- 1


x is the concentration of microbial biomass

t is the time, in hours

µ is the specific growth rate, in hour -1

on integration, equation (1) gives

𝒅𝒅 = 𝒅𝒅 𝒅µ𝒅 ------------------------- 2


𝒅𝒅 is the original biomass concentration

𝒅𝒅 is the biomass concentration after time interval, t hours

During the exponential phase, the organism is growing at its maximum specific growth rate,
µ𝒅𝒅𝒅 for the prevailing conditions.

Equation 2 predicts that growth will continue indefinitely. However, growth results in the
consumption of nutrients and the excretion of microbial products. Thus after a certain time the cell
growth rate will decrease until growth ceases. The cessation of growth may be due to the
depletion of some essential nutrient in the medium when there is limitation in substrate.

The decrease in growth rate and the cessation of growth due to the depletion of substrate may
be described by the relationship between µ and the residual growth-limiting substrate as follows:

µ𝒅𝒅𝒅 𝒅
𝒅𝒅+ 𝒅


µ𝒅𝒅𝒅 = maximum growth rate

s = residual substrate concentration

𝒅𝒅 = substrate utilization constant

Figure 4.1: The graph showing the relationship between the parameter of the Monod equation.

The stationary phase in batch culture is the point where the growth rate has declined to zero. In
the other word the growth rate is equivalent to the death rate. The cell death is might due to the
nutrient limitations due to their incorporation into cells during log-phase growth or a build-up of
toxins due to their release of fermentation products also during log-phase growth.

The death phase is the result of the inability of the bacteria to carry out further reproduction as
condition in the medium become less and less supportive of cell division. The nutrient is extremely
insufficient for the growth of the microorganism. Eventually, the number of viable bacterial cells
begins to decline at an exponential rate. Industrial fermentation is usually interrupted at the end
of the exponential growth phase or before the death phase begins.

Figure 4.2: Growth curve of microorganism based on cell number analysis


 Microbe: Escherichia Coli

 Shake flask (250mL flasks and 1000 mL flasks)
 Eppendorf tubes / falcon tube (1.5 mL)
 Cuvettes (spectrophotometer)
 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


(i) Preparation of media

a) Terrific Broth (TB) preparation

1. The recipe as stated at the bottle is followed.
2. The media is autoclaved at 121 0C 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

b) Luria Bertani (LB) preparation

1. The recipe as stated at the bottle is followed for LB media.
2. A certain amount of phosphate buffer is added to give a specific pH which is pH 7.
Refer to table below.

3. Glucose solution is prepared and it should be autoclaved separately before cooled
down in a different bottle.
4. Distilled water is autoclaved which will be used for filling up volume to the needed
volume prepared.
5. When both solutions are cooled, then only both solutions are added.

Table 6.1: Buffer recipe according to 50 mM Buffer Strength

No Phosphate buffer Concentration
1 K2HPO4 8 g/l
2 KH2PO4 3 g/l

(ii) Preparation of cell culture

a) Seed culture preparation (inoculum)

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 inoculum 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.

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 be
15mL 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:
Table 6.2: Sampling
No. Sample Name Volume (uL) Use for
1 OD 1000 Optical measurement using spectrophotometer
2 CDW 1000 For Cell Dry Weight measurement
3 S 1500 Glucose Analysis

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 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.

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

1. Dried centrifuge tubes is weighted and noted 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. The supernatant is taken out and washing with distilled water and centrifuging may be
5. The centrifuge tube is dried(left with biomass only) in oven at 80 oC for overnight
6. The dried centrifuged tubes is left in desiccator for half an hour.
7. The centrifuge tube weighted and note this as final mass (with biomass = Cell Dry
Cell Dry Weight = Final mass – Initial mass

vi) 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).


Luria Bertani Results

Seed Culture Preparation (Inoculum)

Initial OD : 1.278 (12.40pm)
Final OD : 2.532 (4.40pm)

Table 7.1: Shows The Data For Luria Bertani (LB)

No Time OD M1 M2 X ln X S μ Ln (X/X0)
(h) (nm) (g) (g) (g/L) (g/L) (g/L) (1/h)
1 0 0.188 1.084 1.031 -26.5 - 8.71 - 0.000
2 1 0.189 1.076 1.085 4.5 1.504 8.82 - -
3 2 0.307 1.076 1.243 83.5 4.401 8.70 2.897 -
4 3 0.806 1.097 1.091 -3 - 7.92 - -2.179
5 4 0.853 1.103 1.095 -4 1.386 8.09 - -1.891
6 6 0.655 1.098 1.098 - - 5.79 - -
7 8 0.757 1.097 1.078 -9.5 - 5.72 - -1.026
8 10 0.783 1.093 1.093 - - 4.83 - -
9 12 0.802 1.098 1.109 5.5 1.705 4.21 -0.2695 -
10 14 0.282 1.104 1.155 25.5 3.239 3.89 0.767 -
11 16 0.406 1.089 1.091 1 0 3.47 -1.609 -
12 18 0.409 1.098 1.104 3 1.099 3.30 0.549 -
13 20 0.498 1.113 1.095 -9 - 2.90 - -1.080

OD – Absorbance Optical Density
M1 – Empty Centrifuge
M2 – Dried Centrifuge Tube + Sample
X – Cell Dry Weight Concentration
S – Substrate

Graph of Absorbance (nm) against Time



Absorbance 0.6

(nm) 0.4


0 5 10 15 20 25

Figure 7.1: The growth curve of E.coli plotted using absorbance optical density.

ln X against Time
ln X 2.5
0 5 10 15 20
Time (h)

Figure 7.2: Graph of ln X against time which is plotted to construct the growth curve of the cell
by using cell dry weight

By referring Figure 7.2, it show that the exponential phase may be between at 1th and 14th hour.
This is because at 1st to 2nd hour at 12th and 14th hour shows an increasing data. Thus, the data
between one of this period will be used to find the value µmax. I take period of 1st to 2nd hour.

ln X/Xo against Time

0 5 10 15 20 25


y = 0.0554x - 2.0291
-1 R² = 0.5576
ln X/X0


Time (h)

Figure 7.3: Graph of ln X/X0 against time is plotted to find the value of µmax

From Figure 7.3, the value of µmax which is equal to the slope of the graph is 0.0554.

Thus, the doubling time is 12.51 h.

Graph Of Growth Rate Against Substrate Concentration


-0.5 0 1 2 3 4 5 6 7 8 9 10

Figure 7.4: Graph of growth rate, μ against substrate concentration, S

From Figure 7.4 we cannot find the Ks we lack the value of μ thus make the graph uncomplete.
The value of Ks can be determined if the graph complete by using ½ μmax. By using Monod
Equation Ks value can be determined.

Terrific Broth Results

Seed culture preparation (inoculum)

Initial OD : 0.168
Final OD : 1.203
Table 7.2: Absorbance value at 600 nm obtained using Terrific Broth as medium
No. Time (h) Absorbance Optical
Density (nm)
1 0 0.191
2 1 0.442
3 2 0.763
4 3 1.541
5 4 0.888
6 6 0.991
7 8 0.994
8 10 1.110
9 12 1.103
10 14 1.129
11 16 1.526
12 18 1.178
13 20 1.068

Graph of Absorbance (nm) against Time
Absorbance 0.8
(nm) 0.6
0 5 10 15 20 25

Figure 7.5: The growth curve of E.coli plotted using absorbance optical density.

Table 7.3: Results of Cell Dry Weight (CDW) obtained using Terrific Broth as medium

No. Time Empty Empty tube + Cell dry Cell mass ln x ln X/X0
(h) tube + sticker + cell weight (g) concentration,
sticker (g), (g), m2 (m2 – m1) X (g/L)
1 0 1.10991 1.10 0.0009 0.45 -0.7985 0.000
2 1 1.1030 1.11 0.0070 3.50 1.2528 2.0513
3 2 1.0946 1.09 -0.0046 2.30 0.8329 1.6314
4 3 1.0861 1.09 0.0039 1.95 0.6678 1.4663
5 4 1.1064 1.11 0.0036 1.80 0.5878 1.3863
6 6 1.1021 1.11 0.0079 3.95 1.3737 2.1722
7 8 1.0983 1.11 0.0117 5.85 1.7664 2.5649
8 10 1.1032 1.11 0.0068 3.40 1.2238 2.0223
9 12 1.1106 1.13 0.0194 9.70 2.2721 3.0707
10 14 1.1005 1.11 0.0095 4.75 1.5581 2.3567
11 16 1.0920 1.10 0.0080 4.00 1.3863 2.1848
12 18 1.1029 1.11 0.0071 3.55 1.2669 2.0655
13 20 1.1025 1.11 0.0075 3.75 1.3218 2.1203

ln X against Time

ln X


0 5 10 15 20 25
Time (h)

Figure 7.6: Graph of ln X against time which is plotted to construct the growth curve of the cell
by using cell dry weight
By referring Figure 7.6, it show that the exponential phase may at between at 4th and 12th hour.
Thus, the data between that particular times will be used to find the value µmax.

ln X/Xo against Time

y = 0.1997x + 0.8006
R² = 0.9213

ln X/X0
1.5 Y-Values

1 Linear (Y-Values)


0 5 10 15
Time (h)

Figure 7.7: Graph of ln X/X0 against time is plotted to find the value of µmax

From Figure 7.7, the value of µmax which is equal to the slope of the graph is 0.1997.

Thus, the doubling time is 3.4709.


Cell dry weight, X

X= final weight – initial weight

Volume of Sample

= 1.085 g – 1.076 g
= 0.009 g
Growth Rate, μ
μ = ln x1 – ln x0
Time1 – Time0

= ln 83.5 – ln 4.5

= 2.897 1/h

Yield of substrate, (Y𝒅)

𝒅 ∆𝒅
Y =- (g cells/ g substrate)
𝒅 ∆𝒅

- 25.5 – 5.5 = 62.5 g cells/ g substrate

3.89 – 4.21

Saturation constant (Ks)

μg = μmax . S
Ks +S
No example due to results error


Microorganism 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.
This experiment is carried out to study the kinetic growth of microorganism. E.coli is selected as
the cell and being cultivated inside a shake flask. We used the growth of microorganism in shake
flask which is a simple method of fermentation. The nutrients for the microorganism are being
supplied by the media which contain the carbon sources. In this experiment we used Luria Bertani
(LB) and Terrific Broth (TB) as media. The flask is shaken during the cultivation to ensure constant
mixing of the cell and the media therefore increase the homogeneity between these two and also
to provide aeration for the cells. The culture is gone through the fermentation process for 20 hours.

Within that period, the biomass/cell sample is taken out for every 1 hours from 0 hour to 4 hour
and continued every 2 hour from 4 hour to 20 hour. This is done to analyze the concentration of
the cell (g/L), the cell dry weight and the glucose concentration.

In order to analyze the concentration of the cell inside the flask, absorbance reading for the optical
density is taken from the spectrophotometer. The higher the absorbance reading means higher
number of cell presence inside the flask at a particular time. As for this experiment, the
absorbance reading for LB is increase from the beginning of the experiment until the 6th hour and
decrease slightly until the 8th hour then increase back before decrease until 20th hour. It can be
explained that the number of cell increase and decrease inconsistently throughout the cultivation.
In the other hand, the decrease in cell number in and 14th hour indicating that the cell growth
has been interrupted maybe because there are contamination occur to the sample. The
absorbance reading for TB shows increasing until 10th hour then it decrease a bit before increase
until 18th hour. For TB media, the results shows better reading maybe there are no contamination
occur during conducting the experiment. When there is other microorganism in the culture, the
growth will be interrupted thus the growth of E. coli started to slow down. When there are different
microorganism during the growth they will compete each other to keep living. Only the strong
microorganism can 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. We can say the absorbance
experiment is fail due to inconsistence result.

In cell cultivation, the cells themselves need food or carbon sources like glucose for growth. In
batch fermentation for example in this experiment, the glucose can be the limiting factor for the
cell growth or we called it as substrate limiting growth. For this condition, the Monod equation can
be used to predict the growth rate and the cell concentration inside the shake flask. In addition,
the glucose concentration can be known by testing the cell sample into the glucose analyzer and
the direct glucose concentration can be obtained. The LB media experiment, there are glucose
concentration in there and the reading was obtained by using glucose analyzer. From the results
of substrate concentration, we manage to get what we suppose to get which is the reading will
decrease against time. Although the starting reading is lower than the second reading, the third
until the last reading shows decreasing.

For the graph ln X against time, we also fail to get the estimated result. This graph is plotted to
determine the gradient of exponential phase which is the μmax. The estimated exponential phase
for LB media is from 1st hour until 14th hour. The graph shows decreasing at 2nd hour might be
because of contamination. The reason why I estimate the exponential growth until 14th hour
because the growth occur until that hour. The µmax for this period is 0.0554. For TB media, the
estimated exponential phase may at between at 4th and 12th hour thus, the data between that
particular times will be used to find the value µmax which is 0.1997. The graph below shows the
result that we should achieved but we fail due to so many error.

Figure 9.1: Graph the number of cell against time

Another analysis that can be performed to analyze 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
centrifuged tube. Before that, the empty centrifuged tube is weighted. Then the tube is the being
centrifuged to separate the supernatant with the cell. For LB media, the supernatant is separate
and will be used for glucose analysis but for TB media, the supernatant is separated and removed.
The remained cell is washed with distilled water and centrifuged again before being dried inside
an oven for 24 hours. The dry cell weight with tube is finally taken and the result will be subtract
with the weight of initial empty tube to know the weight of the cell that present at particular time
during the cultivation. In this experiment, the cell dry weight should increased from 0 th hour until
the 20th hour. Unfortunately, we did not manage to get like that because the electronic weight
balance is not accurate. We weight each tube for two times but still get different reading. The
worst part comes when the final reading of cell dry weight with tube is smaller than the initial
reading of empty tube. This will give negative reading thus it is impossible for us to produce a
good graph. When we get negative reading for cell dry weight, we cannot calculate the growth
rate and this happen for LB media.


After conducting the experiment we can ensure that, E.coli is suitable to be fermented inside a
shake flask and it is a simple method to investigate the growth kinetics of the microorganism.
Knowledge of microbial growth kinetics is essential to determine when to harvest the culture for
different purposes. Although we fail to achieve the objectives due to some errors during
conducting the experiment, we try to learn our mistake for next experiment.
Microorganism will go through several phases in their growth, several analyses on the cell
need to be done to know the growth kinetics of the cell and the duration for each phase. This
includes the cell concentration, glucose concentration and also the cell dry weight analyses. This
method can be done in the laboratory before the fermentation or the cultivation of microbes in
large scale is performed. Growth kinetics deals with the rate of cell growth and how it is affected
by various chemical and physical conditions. During the course of growth, the cells is continuously
changing and adapting itself in the media environment, which is also continuously changing in
physical and chemical conditions.
In conclusion, the microbial culture in batch culture system that is shake flask system goes
through a lag phase, exponential growth phase, decelerating growth phase, stationary phase and
sometimes the death phase depends on the end product desired. The substrate concentration in
the culture medium and growth parameters, such as glucose concentration changes
correspondingly throughout the growth phases. Thus, the physiology of the microorganisms is
always in a transient stage, subjected to a continually changing culture conditions. Consequently,
product formation is confined to a certain period of cultivation, for example antibiotics would only
be produced in the decelerating and stationary growth phases.
The batch culture system is still widely used in certain industrial processes for example
brewery industry because of its easy management of feed stocks. These advantages allow the
use of unskilled labor and low risk of financial loss. Low level of microbial contamination in
fermented products is at time tolerable, as long as the microbial contaminants are not pathogenic
and do not alter the desired properties of the product, such as taste, color and texture.


– Aseptic technique must be practised when handling biomass concentration to avoid any
– Cuvette must be wiped cleanly to prevent any scratch that would affect the
spectrophotometer reading during protein test.
– Disinfect the work area with 70% alcohol before handling the culture.
– Dispose of all contaminated materials in appropriate containers.
– The cap of the viral must be opened to fasten the drying process of the biomass in the
– The supernatant of cell concentration should be taken out carefully without any taking out
of the biomass.
– This experiment must be carried out under the laminar flow to prevent any contamination
to the culture.
– Wash hand after handling the culture.


Ardestani, F., (2011). Investigation of the Nutrient Uptake and Cell Growth Kinetics with Monod
and Moser Models for Penicillium brevicompactum ATCC 16024 in Batch Bioreactor.
culture, Iranica Journal of Energy and Environment (IJEE), 2(2): 117-121

Crueger W and Crueger A., (1990). Biotechnology: A Textbook of Industrial Microbiology. Sinauer
Associates, Sunderland Massachusetts.

Kovárová-Kovar, K., & Egli, T. (1998). Growth Kinetics of Suspended Microbial Cells: From
Single-Substrate-Controlled Growth to Mixed-Substrate Kinetics. Retrieved on October 4,
2015 from http://mmbr.asm.org/content/62/3/646.full

PF Stanbury and A Whitaker.,1984. Principles Of Fermentation Technology. Pergamon Press


Figure 13.1: Result of the experiment after being Figure 13.2: After being heated, the sample
approved is being cooled inside a desiccator.

Figure 13.3: Glucose analysis machine model Figure 13.4: Incubator Shaker
YSI 2700