Professional Documents
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Lab Report
kazkaskazkasako
kazkaskazkasako@kazkaskazkasako.lt
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GRADE SIGNATURE
International University Bremen October 31, 2005
School of Engineering and Science
Introduction
The main goal of the experiments outlined below was to isolate and iden-
tify the unknown bacterium. In addition, some of these experiments were
performed with Escerichia coli and Bacillus subtilis as well and the growth
in batch culture was performed only with E. coli. The unknown bacterium
was most exactly identified by the sequencing of the 16S rRNA of this bac-
terium and comparing this sequence with known sequences in the data bank.
But other experiments should have complemented the sequencing results by
providing the form, gram staining, antibiotics resistance and the nutrient
preference of this bacterium. Finally, it was obtained that the bacterium be-
longs to the family Micrococcaceae but exact genus or spieces could not be
determined because two genuses and species from this family had the most
similar 16S rRNA sequences.
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Results
Acquiring samples and choosing unknown bacterium
The first sample was taken by pressing the e2 coin into the center of the agar
under sterile conditions and then the agar plate was incubated for 6 days.
There was observed only one colony of fungus in the periphery of the agar
plate but no colonies of bacterium in the plate and therefore the unknown
bacterium was chosen from the sample which was obtained by Ghimire K.
This sample was obtained by pouring some Multivitamin juice from the Ara-
mark juice machine in Krupp college servery on the agar plate and after a
short time the juice was removed and the plate was closed. Then it was
incubated for several days. The picture of the plate of the sample is shown
in the figure 1 but colonies are bigger than just after the incubation and
some other colonies are present because the photo was taken 1 week after
the incubation.
Two different types of colonies were visible on the agar plate: the dark
yellow and the yellowish colonies. The yellowish colonies were chosen for
further investigation. The dark yellow colonies were slimy and prevalent
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on the agar plate while the yellowish ones were forming just 2 colonies on
the plate. When the yellowish colony was viewed under the microscope at
1000 total magnification it contained only one species of bacterium which
are further referred to as the unknown bacterium. This bacterium was in the
form of tetracocci and seemed greenish under the microscope. The colony
which was investigated under the microscope was restreaked.
Till the third lab course day the restreaked colonies have grown and
merged and the photo of them was taken which is shown in the figure 3.
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Figure 3: The photo of the yellowish unknown bacterium
The colony shape was round and the colony margins were smooth and
the colony texture was also smooth.
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Table 1: OD600 of the flask with LB broth inoculated with E. coli
Time (min) OD600
0 0.043
30 0.068
50 0.124
60 0.161
70 0.235
80 0.233
90 0.267
100 0.319
110 0.361
120 0.390
140 0.472
160 0.572
180 1.685
200 0.735
220 0.735
240 0.777
250 0.789
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Figure 4: OD600 of inoculated LB broth with E. coli dependance on time
The table 1 was changed so that in the place of the OD600 value the
logarithm to the base 10 of the OD600 value was taken and the results were
presented in the table 2. This is equivalent to presentation in half-logarithmic
manner the only difference is that the Log(OD 600) values are negative. Such
representation was chosen because it is easier with a program to find the best
fit line to the data and the slope.
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Table 2: Log(OD600 ) of the flask with LB broth inoculated with E. coli
Time (min) Log(OD600 )
0 -1.367
30 -1.167
50 -0.907
60 -0.793
70 -0.629
80 -0.633
90 -0.573
100 -0.496
110 -0.442
120 -0.409
140 -0.326
160 -0.243
180 -0.164
200 -0.134
220 -0.115
240 -0.110
250 -0.103
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Figure 5: Log(OD600 ) of inoculated LB broth with E. coli dependance on
time
Finally, only the points which make line were taken to calculate the slope.
These points are presented in the table 3.
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Table 3: Log(OD600 ) measurements which make line
Time (min) Log(OD600 )
60 -0.793
80 -0.633
90 -0.573
100 -0.496
110 -0.442
120 -0.409
140 -0.326
160 -0.243
180 -0.164
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Figure 6: Log(OD600 ) of inoculated LB broth with E. coli linear dependance
on time
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The OD600 values of the dilutions 10−3 , 10−4 , 10−5 , 10−6 , 10−8 are too big
because they should be zero and this could be due to the error range of
spectrophotometer. CFU means colony forming units.
Table 4: The OD600 values and CFU count of the dilution series
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Table 5: The OD600 and CFU/ml values of different groups
group OD600 CFU/ml
A 1.18 1.2*108
Q 0.96 3.4*108
H >1 5.2*107
K 0.45 9.4*107
D 0.48 5.6*107
P 0.79 9.8*107
W 0.46 3.2*108
E 0.85 3.1*108
N 1 4.8*108
T 0.75 2.3*108
When these values are plotted they are in a distribution which has no
correlation. So the calibration is not possible.
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Table 7: Resistance of unknown bacterium to different concentrations of
nalidixic acid
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Figure 7: Inhibition radius dependance on Log(Concentration of Nalidixic
acid) for unknown bacterium
From the best fit line to the graph the y-axis interception (c1 ) and the
slope (m1 ) are obtained:
c1 = −14.5 (mm)
m1 = 26.3 (mm)
E. coli was also resistant to that nalidixic acid concentration so the exper-
iment was repeated with huger concentrations of nalidixic acid. The results
are presented in the table 8.
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Table 8: Resistance of E. coli to different concentrations of nalidixic acid
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Figure 8: Inhibition radius dependance on Log(Concentration of Nalidixic
acid) for E. coli
From the best fit line to the graph the y-axis interception (c2 ) and the
slope (m2 ) are obtained:
c2 = 4.3 (mm)
m2 = 13.7 (mm)
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Polymerase chain reaction (PCR) and agarose gel elec-
trophoresis
The product of polymerase chain reaction was confirmed by gel electrophore-
sis. The photo was taken of the gel and it is shown in the figure 9. C- means
negative control, C+ means positive control (E. coli), AK means the run of
PCR product of the unknown bacterium, KG and JK are the runs of PCR
products of other bacteria and M means the run of commercially available
DNA standard marker. It is clearly seen that the amount of the produced
DNA by PCR is huger than the amount in the marker.
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Figure 9: Agarose gel electrophoresis of the PCR products
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Discussion
Acquiring samples and choosing unknown bacterium
The dark yellow bacterium colonies were prevalent on the agar plate over
the yellowish ones because the dark yellow bacterium was more spread in
the Multivitamin juice at room temperature or just because they had slimy
colonies which were prone to stick to the agar plate more than the yellowish
ones. The second possibility maybe the case because it was hard to take a
colony of the dark yellow bacterium by a needle because it sticked better to
the agar than to the inoculation needle while the yellowish ones were easily
taken with the needle.
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Growth of Escherichia coli in batch culture
The measured generation time of E. coli (g = 1.00 h) is several times longer
than the theoretical one because the flask inoculated with E. coli was not all
the time in the warm incubator because the flask was taken out for several
minutes to take 0.5 ml sample for OD600 measurements and so the flask was
cooled. Also one point in the graphs and data was disregarded because it
introduced a small peak.
References
[1] Ullrich, M. 2005: Handling of microorganisms, lab manual, IUB
[2] Ullrich, M. 2005: Microscopy, lab manual, IUB
[3] Ullrich, M. 2005: Gram staining procedure, lab manual, IUB
[4] Ullrich, M. 2005: Growth of Escherichia coli in batch culture, lab
manual, IUB
[5] Ullrich, M. 2005: Antibiotics resistance assay I, lab manual, IUB
[6] Ullrich, M. 2005: Polymerase Chain Reaction (PCR), lab manual, IUB
[7] Ullrich, M. 2005: DNA agarose electrophoresis, lab manual, IUB
[8] Ullrich, M. 2005: Antibiotics resistance assay II - Determination of MIC,
lab manual, IUB
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[9] Ullrich, M. 2005: The API 20E System for Identification of Unknown
Bacteria, lab manual, IUB
[10] NCBI BLAST <http://www.ncbi.nlm.nih.gov/BLAST/> (visited
October 30, 2005)
[11] NCBI, Micrococcaceae
<http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi
?mode=Undef&id=1268&lvl=3&keep=1&srchmode=1&unlock> (visited
October 30, 2005)
[12] Wikipedia, Micrococcaceae
<http://en.wikipedia.org/wiki/Micrococcaceae> (visited October 30, 2005)
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