Investigating the principle of cell growth.

containing one tube of nutrient broth (pre-

Introduction inoculated with E.coli)
The aim of the practical was to determine the Metal inoculating loop
growth rate of E. coli growing at three different Cuvettes
temperatures under batch conditions. E. coli is a
type of bacteria that lives in intestines of animals, Spectrophotometer
most types of E. coli are harmless but some can Pipette 1000µm
cause death[ CITATION Eco \l 2057 ] . The Pipette tips
practical consisted of three inoculated nutrient Petri dish with E. coli on agar
broths cultured under three different temperatures.
Bunsen burner
One at 37oC, as the optimal body temperature for a
human, is around 37.5oC [ CITATION Che \l Igniter
2057 ]. Another at a lower temperature at 20oC, Waste container with Virkon solution
which is approximately room temperature. The
final temperature was an elevated temperature at The bench was wiped down with a solution of
45oC. With the use of a spectrophotometer set at virkon to allow for an aseptic condition. A loop
600nm the scattering of lights caused by the was sterilised using a Bunsen burner, then cooled
turbidity of the solution is measured in optical by taping it at the unused part of the agar plate. The
density, OD[ CITATION Gor \l 2057 ]. loop was then used to pick up a colony from the
agar plate and was mixed into the nutrient broth.
To calculate the cell growth rate, there was a series The nutrient broth was inoculated with E. coli and
of variables to consider, NO, the number of cells at the loop was re-heated to be sterilised.
time 0, Nt, the number of cells at a given time, r,
the intrinsic growth rate that represents the balance Three Tubes which already have been inoculated
between cell division and cell death and t, time. with E. coli were used for the water baths at
Using a differential equation, r can be given as: temperatures of 20oC, 37oC and 45oC and were
labelled correspondently.
dN
r= =μN (1) Using the control nutrient broth as a blank sample
dt to calibrate the spectrometer, the desired
Where r is the change in cell population number wavelength of light was set to 600nm.
with time and µ, is the specific growth rate and N is
the concentration of cells. By separating out the 2000µl of the inoculated nutrient broth was
variable the equation can be re-written as follows: pipetted (the lid of the nutrient broth was flamed
before being opened and when the lid was closed
Nt=N o e μt (2) then the nutrient broth was returned to its labelled
By taking the natural logs of both side of the water bath) into a clean cuvette using a
equation, the equation can be re-written in the form micropipette (two pipettes were used as one pipette
of an equation for a straight line (y=mx+c) as held up to 1000 µl of solution and each time the
follows: broth was pipetted the pipettes were ejected into
waste beaker filled with virkon solution). When
ln Nt =ln N o + μt (3) working with the nutrient broth it was kept as close
Where y = lnNt, m (the gradient of the slope) = µ, x as possible to the Bunsen flame so there was no
= time, t and c (the y-intercept) = lnNo. contamination brought into the solution.

Materials and Method The sample was inserted into the spectrometer and
a reading was recorded into a suitable table (the
Table 1: Shows the materials used for the cuvette was discarded into the waste beaker).
experiment.
This was repeated every 20 minutes 20 times then
Materials again 24 hours after the reaction started.
Two tubes containing nutrient broth
Access to water baths (20°c, 37°c, 45°c)- each Result
Table 1: Shows the change in absorbance of cells 0.45
as time increases. 0.4
Time/min Absorbance OD600 0.35 Lag Phase
  20°c 37°c 45°c
0.3
0 0 0.001 0.001 Exponenti
0.25 al Phase

OD 600
20 0.001 0.002 0.003
0.2 Decelerati
40 0.001 0.002 0.002 on Phase
0.15
60 0.003 0.002 0.003
Stationary
80 0.003 0.003 0.009 0.1
Phase
100 0.01 0.008 0.01 0.05
Decline
120 0.016 0.023 0.011 0 Phase
140 0.025 0.021 0.021 0 50 100 150 200 250 300 350 400
Time/minutes
160 0.026 0.08 0.065
180 0.029 0.116 0.091 Figure 2: Shows a graph of absorbance versus time
200 0.035 0.105 0.095 for 37OC with the stages of bacterial growth.
220 0.036 0.127 0.12
240 0.04 0.159 0.148
0.3
260 0.03 0.208 0.153
280 0.042 0.322 0.158 0.25
300 0.04 0.421 0.161
0.2
320 0.05 0.425 0.171 Lag
OD 600

0.15 Phase
340 0.058 0.41 0.21 Exponenti
360 0.07 0.405 0.245 0.1 al Phase
Decelera
380 0.071 0.44 0.244 0.05 tion
Phase
400 0.087 0.439 0.242 Decline
0
1440 (24hrs) 0.334 0.542 0.256 Phase
0 50 100 150 200 250 300 350 400
0.1 Time/minutes
0.09 Lag Phase Figure 3: Shows a graph of absorbance versus time
0.08 for 45OC with the stages of bacterial growth.
0.07 Exponenti
al Phase
0.06 It was assumed that the exponential phases in each
OD 600

0.05 graph produced a straight line.

0.04
0.03 Using equation 3, the line of the equation for the
0.02 exponential phase for figure 1, 2 and 3 was given
0.01 as y = 0.0002x - 0.011, y = 0.0016x - 0.192, and y
0 = 0.0008x - 0.0605 respectively hence the cell
0 50 100 150 200 250 300 350 400
Time/minutes growth rate for temperatures 20oC, 37oC and 45oC
Figure 1: Shows a graph of absorbance versus time is 0.0002 min-1, 0.0016 min-1 and 0.0008 min-1
for 20OC with the stages of bacterial growth. respectively.

Discussion
The growth of bacteria acts in 5 phases, lag phase,
where the bacteria culture has not produced any
cells, exponential phase, where the bacteria is
producing cells at its fastest rate via catabolic and
anabolic reactions. Deceleration phase, where the
rate of cells being produced has started to slow
down, stationary phase, where the net number of References
cells made are zero and finally the decline/ death
phase, where the cells start to denature and die.
With figures 1, 2 and 3 the bacteria cultures show
the different times at which each the cultures have
reached the different phases. It was shown that the
culture incubated at 20oC had the lowest cell grow
rate followed by the 45oC then 37oC with the
highest growth rate. The optimal temperature for E.
coli to grow is 37oC [ CITATION Gad08 \l 2057 ]
thus, the culture in the 37oC water bath should have
had the highest cell growth rate which is supported
by the experimental values. Therefore,
temperatures that are below and above the optimum
temperature should have a lower absorbance rate
after 24 hours of the bacteria being inoculated,
which is shown in table 2 as the absorbance for the
20oC, 37oC and 45oC incubators were 0.334, 0.542
and 0.256 respectively.

Conclusion
Studying the cell growth rate of bacteria at different
temperatures is a good technique to understand
how bacteria cells live in different conditions. As
E. coli is a bacterium that can cause harm to
humans and other animals by adapting to its
environments. By analysing the temperatures at
which the cells grow and die in the batch
conditions, a scientist can optimise conditions of
cultures used in bioreactors for pharmaceuticals.

Chemistry for Biologists: Enzymes. (n.d.). Retrieved from Rsc.org:

http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm

E. coli | FoodSafety.gov. (n.d.). Retrieved from Foodsafety.gov:

https://www.foodsafety.gov/poisoning/causes/bacteriaviruses/ecoli/

Gadgil, M., Kapur, V., & Hu, W. (2008). Transcriptional Response of Escherichia coli to Temperature Shift.
Biotechnology Progress, 21(3), 689-699. doi:10.1021/bp049630l

Gore, M. G. (2000). Spectrophotometry And Spectrofluorimetry, page 4. Oxford University Press.

Questions

You must complete the following questions and

include your answers with your practical write up
as part of your portfolio.

Three cultures of yeast were grown anaerobically at

30°C in flasks containing 1% glucose solution (=
10g/l) until they reached stationary phase. Use the
data in the table below to calculate the following:
 (a) The average OD in the flasks at the start (g) The molar yield of glucose which is also
and the end of the fermentation equal to the stoichiometric coefficient c
respectively. biomass from the equation given earlier on
page 1:
moles biomass produced mol wt . glucose
c= = Y =
0.369+0.391+0.383 moles glucose consumed mol wt . biomass XS
=0.381
3

(b) The average biomass density,Bd, at the end (h) Calculate the percentage of carbon in the
of the fermentation in units of grams dry glucose that ends up in the biomass and
biomass per litre. postulates what happens to the rest of the
carbon contained in the glucose.
Mass 0.381
Bd = = =1.524 ×10−5 g/l
Volume 25 × 1000 6 atoms of C in glucose

9.887 ×10−6 −5
(c) The average biomass density at the start of ¿ ×100=3.99× 10 %
the fermentation in units of grams dry 24.75
biomass per litre assuming that OD is
proportional to biomass concentration.

Average OD at start = 0.0663

Average OD at end = 0.4473

0.0663
Bd =1.524 ×10−5 × =2.259 ×10−6 g /l
0.4473

(d) The grams of biomass produced per litre

during the fermentation.

Final-initial = 1.524x10-5 – 2.259x10-6

= 1.298x10-5 g/l

(e) The biomass yield from glucose assuming

that all the glucose is consumed during the
fermentation:

grams biomass produced 1.298× 10−5 −6

Y XS= = =1.298 ×10
grams glucose consumed 10

(f) The molecular weights of glucose and

biomass using the formula for biomass
from the equation given earlier on page 1.

Glucose= 180 g

CH1.75O0.53N0.18

∴ MW= (1 ×12 ) + ( 1.75 ×1 ) + ( 0.53 ×16 ) + ( 0.1× 14 ) =23.63 g /mol