Bacterial growth curve
by OD
600
and SoloVPE
Biofactory Competence Center
July – September 2019
Done by
Dr. Tanja Buch and Bc. Michaela Rollová
i
Abstract
The increase in the cell size and cell mass during the development of an organism is termed as
growth. It is the unique characteristics of all organisms. The organism must require certain basic
parameters for their energy generation and cellular biosynthesis. The growth of the organism is
affected by both physical and Nutritional factors. The physical factors include the pH, temperature,
Osmotic pressure, Hydrostatic pressure, and Moisture content of the medium in which the organism
is growing. The nutritional factors include the amount of Carbon, nitrogen, Sulphur, phosphorous, and
other trace elements provided in the growth medium. Bacteria are unicellular (single cell) organisms.
When the bacteria reach a certain size, they divide by binary fission, in which the one cell divides into
two, two into four and continue the process in a geometric fashion. The bacterium is then known to
be in an actively growing phase. To study the bacterial growth population, the viable cells of the
bacterium should be inoculated on to the sterile broth and incubated under optimal growth conditions.
The bacterium starts utilising the components of the media and it will increase in its size and cellular
mass.
The dynamics of the bacterial growth can be studied by plotting the cell growth (absorbance)
versus the incubation time or log of cell number versus time. The curve thus obtained is a sigmoid
curve and is known as a standard growth curve. The increase in the cell mass of the organism is
measured by using the Spectrophotometer. The Spectrophotometer measures the turbidity or Optical
density which is the measure of the amount of light absorbed by a bacterial suspension. The degree of
turbidity in the broth culture is directly related to the number of microorganism present, either viable
or dead cells, and is a convenient and rapid method of measuring cell growth rate of an organism.
Thus, the increasing the turbidity of the broth medium indicates increase of the microbial cell mass.
The amount of transmitted light through turbid broth decreases with subsequent increase in the
absorbance value.
This traditional methodology relying on fixed pathlength UV spectroscopy can require several
minutes of stagnant time because of the need for careful sample handling, preparation (base-line
correction), and (in particular) dilutions needed for bringing samples into the spectrophotometer’s
linear range. The doubling time of many bacterial cells is very short. Errors created in performing those
dilutions and baseline corrections can take longer that the doubling time of bacterial cells and can
significantly affect calculated sample optical density.
The SoloVPE is the laboratory implementation of C Technologies, Inc.’s variable pathlength
technology using Slope Spectroscopy methods that are based upon the Slope Spectroscopy Equation
which is fundamentally derived from Beer’s Law. Slope Spectroscopy analysis with variable pathlength
requires no sample preparation, no baseline correction and no dilution of even the most highly
cell-concentrated samples, saving substantial time without changing any other aspect of the assay.
In this work, the bacterial growth curve by two methods during bacterial cells cultivation is
investigated. These are the standard spectrophotometer and the SoloVPE method. For cultivation,
Escherichia coli cells (E. coli K-12 W3110) and YPG medium were used. The bacterial growth curve was
determined and standard spectrophotometer and SoloVPE were compared. Additionally, a correlation
between these two methods was found. This principle allows a fast and cheap method to analyze
bacterial cells samples during bacterial cell cultivation by avoiding baseline corrections and dilutions
and getting accurate results faster and with less effort.
ii
Abbreviations
DI-water = Deionized water
EC = Escherichia coli
OD = Optical density
OD600 = Optical density at wavelength 600 nm
iii
Table of contents
1 Introduction ................................................................................................................................ 1
Theoretical part ...................................................................................................................................... 1
2 Measuring principle .................................................................................................................... 1
2.1 Bacterial growth curve ............................................................................................................ 1
2.2 STD. SPECTROPHOTOMETER ................................................................................................... 2
2.3 SoloVPE .................................................................................................................................... 3
3 Measuring range, limitations and advantages .......................................................................... 4
3.1 STD. SPECTROPHOTOMETER ................................................................................................... 4
3.2 SoloVPE .................................................................................................................................... 4
Practical Part ........................................................................................................................................... 5
4 Experimental plan ....................................................................................................................... 5
5 Results and discussions .............................................................................................................. 5
5.1 STD. SPECTROPHOTOMETER ................................................................................................... 5
5.2 SoloVPE .................................................................................................................................... 6
5.3 Comparison of Standard spectrophotometer and SoloVPE .......................................................... 7
6 Conclusion ................................................................................................................................. 10
7 Sources ...................................................................................................................................... 11
1
1 Introduction
The aim of this study is to detect the bacterial growth curve of E. coli by two methods, such as standard
spectrophotometer and SoloVPE method. The cell-concentration range, which can be analyzed by
those methods, will be tested to show the reasonable statistical certainty and time consumption during
measurements. This allows to find the most suitable method for further bacterial cells analysis during
bacterial cells cultivation.
Theoretical part
2 Measuring principle
2.1 Bacterial growth curve
The growth curve has four distinct phases (Fig 1)
2.1.1 Lag phase
When a microorganism is introduced into the fresh medium, it takes some time to adjust with the
new environment. This phase is termed as Lag phase, in which cellular metabolism is accelerated,
cells are increasing in size, but the bacteria are not able to replicate and therefore no increase in cell
mass. The length of the lag phase depends directly on the previous growth condition of the organism.
When the microorganism growing in a rich medium is inoculated into nutritionally poor medium, the
organism will take more time to adapt with the new environment. The organism will start
synthesising the necessary proteins, co-enzymes and vitamins needed for their growth and hence
there will be a subsequent increase in the lag phase. Similarly when an organism from a nutritionally
poor medium is added to a nutritionally rich medium, the organism can easily adapt to the
environment, it can start the cell division without any delay, and therefore will have less lag phase it
may be absent.
2.1.2 Exponential or Logarithmic (log) phase
During this phase, the microorganisms are in a rapidly growing and dividing state. Their metabolic
activity increases and the organism begin the DNA replication by binary fission at a constant rate. The
Figure 1: Different phases of bacterial growth
2
growth medium is exploited at the maximal rate, the culture reaches the maximum growth rate and
the number of bacteria increases logarithmically (exponentially) and finally the single cell divide into
two, which replicate into four, eight, sixteen, thirty two and so on (That is 2
0
, 2
1
, 2
2
, 2
3
.........2
n
, n is
the number of generations) This will result in a balanced growth. The time taken by the bacteria to
double in number during a specified time period is known as the generation time. The generation
time tends to vary with different organisms. E.coli divides in every 20 minutes, hence its generation
time is 20 minutes, and for Staphylococcus aureus it is 30 minutes.
2.1.3 Stationary phase
As the bacterial population continues to grow, all the nutrients in the growth medium are used up by
the microorganism for their rapid multiplication. This result in the accumulation of waste materials,
toxic metabolites and inhibitory compounds such as antibiotics in the medium. This shifts the
conditions of the medium such as pH and temperature, thereby creating an unfavourable
environment for the bacterial growth. The reproduction rate will slow down, the cells undergoing
division is equal to the number of cell death, and finally bacterium stops its division completely. The
cell number is not increased and thus the growth rate is stabilised. If a cell taken from the stationary
phase is introduced into a fresh medium, the cell can easily move on the exponential phase and is
able to perform its metabolic activities as usual.
2.1.4 Decline or Death phase
The depletion of nutrients and the subsequent accumulation of metabolic waste products and other
toxic materials in the media will facilitates the bacterium to move on to the Death phase. During
this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to die due to
the unfavourable conditions and the death is rapid and at uniform rate. The number of dead cells
exceeds the number of live cells. Some organisms which can resist this condition can survive in the
environment by producing endospores.
2.2 Standard spectrophotometer
This method allows to determine the turbidity or Optical density which is the measure of the amount
of light absorbed by a bacterial suspension. The degree of turbidity in the broth culture is directly
related to the number of microorganism present, either viable or dead cells, and is a convenient and
rapid method of measuring cell growth rate of an organism. Thus, the increasing the turbidity of the
broth medium indicates increase of the microbial cell mass (Fig 2). The amount of transmitted light
through turbid broth decreases with subsequent increase in the absorbance value.
Figure 2: Absorbance reading of bacterial suspension
3
2.3 SoloVPE
By evolving beyond the limitations of traditional fixed pathlength spectroscopy, the SoloVPE method
expanded the mature UV-Vis technique from a 2-dimensional to a 3-dimensional science. Conceptually
simple, but analytically empowering, C Technologies’ variable pathlength solutions (shown in figure 3)
have revolutionized the measurement of concentration by delivering rapid and accurate results while
avoiding costly dilution and background correction steps on the widest range of samples which is all
made possible by the Slope Spectroscopy technique.
Unlike the single value dependence of legacy UV-Vis methods, the data dense slope method
characterizes samples by collecting multiple absorbance data points at several pathlengths to create a
section curve (Absorbance vs. Pathlength plot). Light is transmitted through the sample via an optical
fiber or fibrette (shown in figure 4). Using a step motor to control the pathlengths from 5 µm to 15
mm (depending on a vessel type, that are shown in figure 5) in as small as 5 µm steps. The section
curve is then analyzed in real time to verify linearity in compliance with the Beer-Lambert Law. The
linear region of the section curve is directly proportional to the concentration of the sample based
upon the sample extinction coefficient. Capable of making spectral and fixedpoint measurements at
wavelengths between 190 and 1100 nm and at pathlengths between 5 microns and 15 millimeters,
this relationship allows the SoloVPE system to measure low and high concentrated samples directly
and report concentration results in less than 60 seconds.
Figure 3: Schematic principle of variable pathlength of
SoloVPE method
[12]
Figure 4: Fibrettes - Disposable or reusable Solid core UV transmissive silica
[12]
4
3 Measuring range, limitations and advantages
3.1 Standard spectrophotometer
The OD measuring range found in the literature of the standard spectrophotometer method is
approximately between 0.5 g/L and 1.0 g/L. A wavelength of 600 nm is used to determine the bacterial
cell concentration/optical density. The bacterial cells absorption properties are around absorbance of
1. Because bacterial cells, that absorb at 600 nm, will may not absorb at wavelength higher or lower
than 600 nm, with higher cell-concentrations it leads to an error in the measuring result.
By using wavelength of 600nm, disposable plastic cuvettes must be used to avoid any interferences
with the cuvette during the measurement. Limitations of this method are, as already mentioned, the
absorption properties of the bacterial cells and the interferences with cultivation medium, in other
words, the need of dilutions and baseline corrections. However, this method is simple to use and non-
destructive which allows a fast and reliable measure of bacterial cells growth.
3.2 SoloVPE
The measure range of the SoloVPE method for bacterial cell cultivation is not determined. The great
advantage of SoloVPE method is that still OD
600
and Beer’s Law is applied, however it can keep chosen
specific concentration or extinction coefficient fixed by changing the pathlength to take linear
measurements. Capable of making spectral and fixed-point measurements at wavelengths between
190 and 1100 nm at pathlengths between 5 µm and 15 mm, the SoloVPE Solution is adaptable to a
wide range of sample types and concentrations. Finally, it can be said that this method allows a fast
analyze of a high range of proteins. However, like all methods, the SoloVPE method has some
disadvantages, these are mainly the beginning investment price and consumability of fibrettes and
vessels.
UV quartz vessels
Disposable UV plastic vessel (small)
Figure 5: Vessels - Disposable or reusable vessels for SoloVPE method
[12]
5
Practical Part
4 Experimental plan
To measure the growth curve for E. coli, two samples (one from each shake flask) are taken every 30
minutes in order to find the absorbance/optical density (OD) or Slope absorbance at wavelength 600
nm and to test and compare the results detected by two methods, such as standard
spectrophotometer and SoloVPE. To compare the results, E. coli K-12 W3110 cells and YPG medium
(glucose added into the medium before autoclaving) were used. The cultivation was held in an
incubator set to 37 °C and 150 rpm.
The comparison study is divided into three parts. The first part examines the growth curve detected
by standard spectrophotometer method. In the second part, the growth curve, determined by Slope
absorbance using SoloVPE method is investigated. The third part of this study compares results
detected by standard spectrophotometer and SoloVPE method and examines the differences between
of these methods. Furthermore, the correlation between the two methods is determined.
4.1 SoloVPE settings – Quick Slope
- Slope Mode: Fixed
- … (next to the Slope Mode): - Start PL: 3 mm
- Step PL: 0,15 mm
- Sample vessel: SV1-Small
- Wavelength: 600 nm
- Extinction coefficient: 1 mL/mg.cm
5 Results and discussions
5.1 Standard spectrophotometer
Time [h]
Absorbance
Average
absorbance
Standard
deviation
Shake flask 1
Shake flask 2
0
0,041
0,039
0,0400
0,001414
0,5
0,059
0,054
0,0565
0,003536
1
0,091
0,097
0,0940
0,004243
1,5
0,202
0,183
0,1925
0,013435
2
0,412
0,394
0,4030
0,012728
2,5
1,160
1,090
1,1250
0,049497
3
1,900
1,830
1,8650
0,049497
3,5
2,530
2,430
2,4800
0,070711
4
3,120
2,880
3,0000
0,169706
5
3,180
3,030
3,1050
0,106066
Table 1: Absorbance/Optical density of the E. coli suspension during bacterial cells cultivation measured by
standard spectrophotometer at wavelength of 600 nm
6
To investigate the OD
600
for higher concentrations of E. coli cells by standard spectrophotometer,
dilutions had to be done. First dilution was prepared after 2.5 hours of cultivation. For all dilutions, a
10-fold dilution was performed.
The repetability of the method was tested by repeating the E. coli cell cultivation in two or three shake
flasks three times. The results are presented in table 2. The standard spectrophotometer method
showed a good repetability.
5.2 SoloVPE
Time [h]
Slope absorbance
Average
absorbance
Standard
deviation
Shake flask 1
Shake flask 2
0
0,01549
0,01536
0,015
0,0001
0,5
0,01566
0,01636
0,016
0,0005
1
0,01564
0,01608
0,016
0,0003
1,5
0,01702
0,01693
0,017
0,0001
2
0,01871
0,01898
0,019
0,0002
2,5
0,02466
0,02314
0,023
0,0011
3
0,03058
0,02985
0,030
0,0005
3,5
0,03683
0,03595
0,036
0,0006
4
0,04223
0,04506
0,045
0,0020
5
0,04481
0,04444
0,044
0,0003
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 1 2 3 4 5 6
Absorbance
Time [h]
Std. spectrophotometer - Average OD
600
Graph 1: Growth curve of the E. coli measured by standard spectrophotometer. Samples taken after 2.5
hour of cultivation were diluted by dilution factor of 10 and then recalculated back to the real OD600.
Table 2: Slope absorbance of the E. coli suspension during bacterial cells cultivation measured by SoloVPE at 600 nm
7
The repetability of the method was tested by repeating the E. coli cell cultivation in two or three shake
flasks three times. The SoloVPE method shows a good repetability.
5.3 Comparison of Standard spectrophotometer and SoloVPE
Additionally, the results measured by two different methods, standard spectrophotometer and
SolovPE, were compared. Both bacterial growth curves had the same shape. A correlation between
these two methods was determined. Furthermore, a recalculation from SoloVPE results to OD
600
(Standard spectrophotometer) results by the linear regression function was done. It was found, that
with larger differences in R
2
(R
2
97,5 % or lower) it is impossible to recalculate results detected by
SoloVPE (Slope absorbances), because the values are very small and the RSD of 2,5 % or higher has a
significant impact on the recalculations.
0,000
0,005
0,010
0,015
0,020
0,025
0,030
0,035
0,040
0,045
0,050
0 1 2 3 4 5 6
Slope absorbance
Time [h]
SoloVPE - Average Slope absorbance
Graph 2: Growth curve of the E. coli measured by SoloVPE.
8
Time [h]
Absorbance
Average
absorbance
Standard
deviation
Shake flask 1
Shake flask 2
0
0,065
0,051
0,058
0,0101
0,5
0,084
0,160
0,122
0,0544
1
0,081
0,130
0,105
0,0342
1,5
0,233
0,223
0,228
0,0070
2
0,419
0,448
0,434
0,0210
2,5
1,073
0,905
0,989
0,1181
3
1,723
1,643
1,683
0,0567
3,5
2,410
2,313
2,362
0,0684
4
3,003
3,314
3,159
0,2199
5
3,287
3,246
3,266
0,0288
y = 0,0091x + 0,0149
R² = 0,993
0,00
0,01
0,01
0,02
0,02
0,03
0,03
0,04
0,04
0,05
0,05
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5
SoloVPE Slope Absorbance
Std. spectrophotometer Absorbance
Comparison of Std. spectrophotometer and SoloVPE
Graph 3: Correlation between results detected by standard spectrophotometer and SoloVPE
Table 3: Recalculation of results detected by SoloVPE (Slope absorbance) during E. coli cultivation into results detected
by standard spectrophotometer (Absorbance/OD
600
) using a function (y = 0,0091x + 0,0149)
9
The repetability and accuracy of determined function was tested by recalculation all SoloVPE results
(Slope absorbances) from three different E. coli cultivations. All measured data were recalculated and
growth curves that were plotted from the recalculated OD
600
showed identical shapes. The function
shows a good repeatability and precision and can be used for recalculation Slope absorbances (SoloVPE
results) to standard absorbances (OD
600
) values, that are detected by standard spectrophotometer.
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0 1 2 3 4 5 6
Absorbance
Time [h]
Recalculated absorbance/OD
600
from Slope absorbance detected by SoloVPE
Graph 4: Growth curve of the E. coli from recalculated results
10
6 Conclusion
It can be concluded that the E. coli cells grew as described in the literature. After 3 hours of cultivation,
due to the strong interferences with high cell-concentrated samples, dilutions had to be performed to
determine the OD
600
by standard spectrophotometer. The SoloVPE method allowed to measure cell
suspension without using any dilutions and baseline corrections.
The difference in growth curves between these two methods can be described by detecting different
parameters by each device. The standard spectrophotometer detects the absorbance, but the SoloVPE
detects Slope absorbance with a unit Abs/mm, that is determined from ten measurements of
absorbances in ten different pathlenthgs. Overall, the to growth curves are very similar in shape.
Additionally, a correlation between these two methods for bacterial cell quantification during bacterial
cell cultivation was found. Furthermore, a SoloVPE results were recalculated into OD600 (standard
spectrophotometer) values by using a function that was determined by linear regression.
Finally, it can be said that SoloVPE method is suitable for measuring the optical density of cells during
bacterial culture. This method is less time consuming and can be recalculated to standard OD600
values by using one simple function.
