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Microbiology

Lab Report

Isolation and Identification of Unknown Microorganism

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.

Materials and Methods


The experiments were conducted according to guidelines in the lab manuals
(Ullrich, 2005 [1], [2], [3], [4], [5], [6], [7], [8], [9]) except taking the sample of
bacterium, determining this bacterium by comparing its 16S rRNA sequence
with the sequences in GenBank and freezing this bacterium in 10% glycerol
solution in water.
The sample under investigation was taken from the e2 coin under sterile
conditions and this was achieved by sticking one side of the coin to the tape
and taking the coin out of the wallet by keeping the two free ends of the tape.
Then the side of the coin without tape was pressed against the agar in the
agar plate and the coin was removed again by taking the ends of the tape
so that agar was not touched by fingers. Finally, the agar plate was closed
and the next day was given to prof. Ullrich in his office. Because no signs of
growth of bacteria were present after 6 days of incubation the other unknown
bacterium was taken under further investigation. This bacterium was from
one type of colonies of the two present in the sample of the Multivitamin
juice from Aramark and this juice sample was taken by Kedar G.
After the sequencing of the 16S rRNA of this unknown bacterium was
done by company the sequence was compared to other known sequences in
website (NCBI BLAST [10]).
The separated bacterium was finally prepared for freezing in a micro re-
action tube in 1 ml of 10% glycerol solution in water under sterile conditions.

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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.

Figure 1: The photo of the plate with Multivitamin juice sample

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.

Gram staining of Eschericia coli and Bacillus subtilis


The next lab course day the gram staining of Escherichia coli and Bacillus
subtilis was carried out. After staining these two species were investigated
under the microscope and E. coli was short gram-negative bacterium while
B. subtilis was long gram-positive bacterium.

The shape of the colonies of the unknown bacterium


and the shape of the unknown bacterium
The unknown bacterium which was incubated in the new agar plate overnight
was also investigated under the microscope and it was confirmed that the
bacterium was forming tetracocci but the bacterium was not round rather
had irregular form or sometimes was a bit elongated in one direction. The
drawing of them is presented in the figure 2.

Figure 2: The drawing of the unknown bacterium from Multivitamin juice


under the microscope, magnification 1000

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.

Gram staining of the unknown bacterium


On the forth lab course day the gram staining of this bacterium was per-
formed and the outcome was that it was gram-negative bacterium.

Growth of Escherichia coli in batch culture


The next day the experiment of the growth of E. coli was performed. The
collected data are presented in the table 1.

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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

The graphical representation of table 1 is given in figure 4.

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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

Table 2 was presented in a figure 5.

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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

Table 3 and linear function were presented in figure 6.

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Figure 6: Log(OD600 ) of inoculated LB broth with E. coli linear dependance
on time

The slope is:


k = 0.0050 min−1
Because the number of cells in 1 ml is directly proportional to the OD600
measurements then the growth rate of E. coli can be calculated from OD600
and it is:
µ = 2.303 × k
µ = 0.0115 min−1 = 0.691 h−1
The generation time is:
g = ln(2)
µ
g = 1.00 h
In order to find the proportionality coefficient between the number of E.
coli cells in 1 ml and the measured OD600 values the calibration experiment
was performed by many groups. Our group worked with medium concentra-
tion of the E. coli suspension and the results are presented in the table 4.

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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

dilution OD600 CFU count Number of cells in undiluted suspension


100 0.457 — —
10−1 0.055 — —
10−2 0.006 many —
10−3 0.002 many —
10−4 0.004 486 4.86*107
10−5 0.002 63 6.3*107
10−6 0.001 19 1.9*108
10−7 0.000 7 7*108
10−8 0.003 — —

Because the calculated number of cells in the undiluted suspension are


quite different so the two most exact ones which are the least diluted are
taken to calculate the number of bacteria per 1 ml.

N = (4.86 ∗ 107 + 6.3 ∗ 107 )/2 = 5.67 (bacteria/ml)

The number of bacteria per 1 ml with corresponding OD600 values from


all groups for calibration are presented in the table 5.

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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.

Antibiotics resistance assays for E. coli, B. subtilis and


the unknown bacterium
The results of the measurements are shown in the table 6.

Table 6: Resistance to different antibiotics


antibiotic E. coli B. subtilis Unknown
Inhibition radius (cm)
Ampicillin 2.5 2.1 4.0
Cloramphenicol 1.9 2.6 0.8
Kanamycin 2.2 2.2 1.7
Nalidixic acid — 2.1 —
Rifampicin 0.8 2.0 4.4

The unknown bacterium was resistant to this nalidixic acid concentration


so the experiment was repeated with huger concentrations of nalidixic acid.
The results are presented in the table 7.

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Table 7: Resistance of unknown bacterium to different concentrations of
nalidixic acid

Concentration of nalidixic acid (mg/ml) Inhibition radius (mm)


20 20
15 16
10 12
5 —
2.5 —
0 —

The results of the unknown bacterium are plotted in a half-logarithmic


graph in the figure 7.

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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)

The formula for minimal inhibition concentration (MIC) is:


c
− m1
M IC1 = 10 1

The calculated value is:

M IC1 = 3.6 (mg/ml)

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

Concentration of nalidixic acid (mg/ml) Inhibition radius (mm)


20 22
15 20
10 18
5 15
2.5 9
0 —

The results of E. coli are plotted in a half-logarithmic graph in the figure 8.

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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)

The formula for minimal inhibition concentration (MIC) is:


c
− m2
M IC2 = 10 2

The calculated value is:

M IC2 = 0.48 (mg/ml)

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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

Biochemical analysis of unknown bacterium


The results of the biochemical analysis of the unknown bacterium are:
1. Positive results: ADH, CIT, URE, VP, GEL.
2. Negative results: ONPG, LDC, ODC, H2 S, TDA, IND, GLU, MAN, INO,
SOR, RHA, SAC, MEL, AMY, ARA.

Sequencing results and comparison of the sequence data


of the unknown bacterium in website
The sequence of 16S rRNA of the unknown bacterium was compared in the
website (NCBI BLAST [10]). The hugest similarity was to Arthrobacter
sp., Micrococcus luteus and Micrococcus sp., Voriovorax sp., Antarctic bac-
terium and Micrococcaceae bacterium among the 18 hits with highest score
and longest nucleotide sequence for comparison. Arthrobacter sp. was in
the 1st and 16th, Micrococcus luteus was in the 2nd, 3rd, 6th, 9th, 12th,
13th and 15th, Micrococcus sp. was in the 4th, 5th, 11th, 17th and 18th,
Voriovorax sp. was in the 8th, Antarctic bacterium was in the 10th and
Micrococcaceae bacterium was in the 14th positions. All these hits had more
than 97% of the nucleotides the same. All of them except Voriovorax sp.
are classified as cellular organisms, Bacteria, Actinobacteria, Actinobacteria
(class), Actinobacteridae, Actinomycetales, Micrococcineae, Micrococcaceae
(NCBI, Micrococcaceae [11]). Arthrobacter sp. and Micrococcus sp. are
genuses from the same family Micrococcaceae. This family contains gram-
positive cocci that inhabit skin and the air (Wikipedia, Micrococcaceae [12]).

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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.

Gram staining of Eschericia coli and Bacillus subtilis


The results of the gram staining were correct but they were only obtained
after several false results because it was realised after several stainings that
the evaporation of water should be done more gently so that the bacteria
would not boil and that during the washing with decolorizer it should be used
as little decolorizer as possible because all the stain can be easily washed out.

The shape of the colonies of the unknown bacterium


and the shape of the unknown bacterium
The shape of the unknown bacterium is compatible with the sequence com-
parison results because it was tetracocci under the microscope and according
to sequence comparison it is cocci but the shape of single bacterium under
the microscope was not spherical maybe because of the limited resolution of
the microscope and distortions.

Gram staining of the unknown bacterium


Gram staining of the bacterium was incorrect because the Micrococcaceae
family contains only gram-positive cocci. This is most probably because
this sample was mixed with another one during gram staining. When the
gram staining was performed three different bacteria were stained on the
same glass. Two of them were assigned incorrect gram staining results, one
gram-positive, another gram-negative. And if these two results are changed
to the opposite ones then the gram staining is in the accord with sequence
comparison results.

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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.

Antibiotics resistance assays for E. coli, B. subtilis and


the unknown bacterium
B. subtilis is susceptable to all antibiotics with specific concentrations applied
at first time while E. coli and the unknown bacterium were not inhibited by
the initial concentration of the nalidixic acid. But during the second exper-
iment the minimal inhibitory concentration of nalidixic acid was measured
and for E. coli it was M IC2 = 0.48 (mg/ml) and for the unknown bacterium
it was M IC1 = 3.6 (mg/ml). So the unknown bacterium is less susceptable
to the nalidixic acid than E. coli.

Polymerase chain reaction (PCR) and agarose gel elec-


trophoresis
Agarose gel electrophoresis showed that the PCR was done correctly because
negative control (C-) was negative and the band of C+ and AK were at the
same position as one of the bands of the marker.

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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