Microbiology

Lab Report
Isolation and Identification of Unknown Microorganism

kazkaskazkasako kazkaskazkasako@kazkaskazkasako.lt

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GRADE
International University Bremen School of Engineering and Science

SIGNATURE
October 31, 2005

Introduction
The main goal of the experiments outlined below was to isolate and identify 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 bacterium 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 belongs 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 reaction 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 Aramark 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 2

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 performed 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 Time (min) 0 30 50 60 70 80 90 100 110 120 140 160 180 200 220 240 250

broth inoculated with E. coli OD600 0.043 0.068 0.124 0.161 0.235 0.233 0.267 0.319 0.361 0.390 0.472 0.572 1.685 0.735 0.735 0.777 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 concentration of the E. coli suspension and the results are presented in the table 4. 10

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 100 10−1 10−2 10−3 10−4 10−5 10−6 10−7 10−8 OD600 0.457 0.055 0.006 0.002 0.004 0.002 0.001 0.000 0.003 CFU count — — many many 486 63 19 7 — Number of cells in undiluted suspension — — — — 4.86*107 6.3*107 1.9*108 7*108 —

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) 20 15 10 5 2.5 0 Inhibition radius (mm) 20 16 12 — — —

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: M IC1 = 10 The calculated value is: M IC1 = 3.6 (mg/ml) E. coli was also resistant to that nalidixic acid concentration so the experiment was repeated with huger concentrations of nalidixic acid. The results are presented in the table 8.
1 −m c 1

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Table 8: Resistance of E. coli to different concentrations of nalidixic acid Concentration of nalidixic acid (mg/ml) 20 15 10 5 2.5 0 Inhibition radius (mm) 22 20 18 15 9 —

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: M IC2 = 10 The calculated value is: M IC2 = 0.48 (mg/ml)
2 −m c 2

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Polymerase chain reaction (PCR) and agarose gel electrophoresis
The product of polymerase chain reaction was confirmed by gel electrophoresis. 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 bacterium 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 grampositive 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 comparison 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 experiment 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 electrophoresis
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: [2] Ullrich, M. 2005: [3] Ullrich, M. 2005: [4] Ullrich, M. 2005: manual, IUB [5] Ullrich, M. 2005: [6] Ullrich, M. 2005: [7] Ullrich, M. 2005: [8] Ullrich, M. 2005: lab manual, IUB Handling of microorganisms, lab manual, IUB Microscopy, lab manual, IUB Gram staining procedure, lab manual, IUB Growth of Escherichia coli in batch culture, lab Antibiotics resistance assay I, lab manual, IUB Polymerase Chain Reaction (PCR), lab manual, IUB DNA agarose electrophoresis, lab manual, IUB Antibiotics resistance assay II - Determination of MIC,

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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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