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Microbiology Lab Report Isolation and Identification of Unknown Microorganism kazkaskazkasako [email protected] —————————— ——————————— GRADE SIGNATURE International University Bremen School of Engineering and Science October 31, 2005
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Isolation and Identification of Unknown Microorganism
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Page 1: Lab Report

MicrobiologyLab Report

Isolation and Identification of Unknown Microorganism

[email protected]

—————————— ———————————

GRADE SIGNATURE

International University BremenSchool of Engineering and Science

October 31, 2005

Page 2: Lab Report

Introduction

The main goal of the experiments outlined below was to isolate and iden-tify the unknown bacterium. In addition, some of these experiments wereperformed with Escerichia coli and Bacillus subtilis as well and the growthin batch culture was performed only with E. coli. The unknown bacteriumwas 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 byproviding the form, gram staining, antibiotics resistance and the nutrientpreference of this bacterium. Finally, it was obtained that the bacterium be-longs to the family Micrococcaceae but exact genus or spieces could not bedetermined because two genuses and species from this family had the mostsimilar 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 ofbacterium, determining this bacterium by comparing its 16S rRNA sequencewith the sequences in GenBank and freezing this bacterium in 10% glycerolsolution in water.

The sample under investigation was taken from the e2 coin under sterileconditions and this was achieved by sticking one side of the coin to the tapeand 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 theagar plate and the coin was removed again by taking the ends of the tapeso that agar was not touched by fingers. Finally, the agar plate was closedand the next day was given to prof. Ullrich in his office. Because no signs ofgrowth of bacteria were present after 6 days of incubation the other unknownbacterium was taken under further investigation. This bacterium was fromone type of colonies of the two present in the sample of the Multivitaminjuice from Aramark and this juice sample was taken by Kedar G.

After the sequencing of the 16S rRNA of this unknown bacterium wasdone by company the sequence was compared to other known sequences inwebsite (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 agarunder 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 agarplate but no colonies of bacterium in the plate and therefore the unknownbacterium 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 ashort time the juice was removed and the plate was closed. Then it wasincubated for several days. The picture of the plate of the sample is shownin the figure 1 but colonies are bigger than just after the incubation andsome other colonies are present because the photo was taken 1 week afterthe incubation.

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

Two different types of colonies were visible on the agar plate: the darkyellow and the yellowish colonies. The yellowish colonies were chosen forfurther 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 onthe plate. When the yellowish colony was viewed under the microscope at1000 total magnification it contained only one species of bacterium whichare further referred to as the unknown bacterium. This bacterium was in theform of tetracocci and seemed greenish under the microscope. The colonywhich 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 Bacillussubtilis was carried out. After staining these two species were investigatedunder the microscope and E. coli was short gram-negative bacterium whileB. subtilis was long gram-positive bacterium.

The shape of the colonies of the unknown bacteriumand the shape of the unknown bacterium

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

Figure 2: The drawing of the unknown bacterium from Multivitamin juiceunder the microscope, magnification 1000

Till the third lab course day the restreaked colonies have grown andmerged and the photo of them was taken which is shown in the figure 3.

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Page 5: Lab Report

Figure 3: The photo of the yellowish unknown bacterium

The colony shape was round and the colony margins were smooth andthe 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. Thecollected data are presented in the table 1.

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Table 1: OD600 of the flask with LB broth inoculated with E. coliTime (min) OD600

0 0.04330 0.06850 0.12460 0.16170 0.23580 0.23390 0.267100 0.319110 0.361120 0.390140 0.472160 0.572180 1.685200 0.735220 0.735240 0.777250 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 thelogarithm to the base 10 of the OD600 value was taken and the results werepresented in the table 2. This is equivalent to presentation in half-logarithmicmanner the only difference is that the Log(OD 600) values are negative. Suchrepresentation was chosen because it is easier with a program to find the bestfit line to the data and the slope.

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Table 2: Log(OD600) of the flask with LB broth inoculated with E. coliTime (min) Log(OD600)

0 -1.36730 -1.16750 -0.90760 -0.79370 -0.62980 -0.63390 -0.573100 -0.496110 -0.442120 -0.409140 -0.326160 -0.243180 -0.164200 -0.134220 -0.115240 -0.110250 -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 ontime

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 lineTime (min) Log(OD600)

60 -0.79380 -0.63390 -0.573100 -0.496110 -0.442120 -0.409140 -0.326160 -0.243180 -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 dependanceon 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× kµ = 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 experimentwas 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 bigbecause they should be zero and this could be due to the error range ofspectrophotometer. 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 suspension100 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 arequite different so the two most exact ones which are the least diluted aretaken 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 fromall groups for calibration are presented in the table 5.

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Table 5: The OD600 and CFU/ml values of different groupsgroup 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 nocorrelation. So the calibration is not possible.

Antibiotics resistance assays for E. coli, B. subtilis andthe unknown bacterium

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

Table 6: Resistance to different antibioticsantibiotic E. coli B. subtilis Unknown

Inhibition radius (cm)Ampicillin 2.5 2.1 4.0

Cloramphenicol 1.9 2.6 0.8Kanamycin 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 concentrationso 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 ofnalidixic acid

Concentration of nalidixic acid (mg/ml) Inhibition radius (mm)20 2015 1610 125 —

2.5 —0 —

The results of the unknown bacterium are plotted in a half-logarithmicgraph in the figure 7.

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Figure 7: Inhibition radius dependance on Log(Concentration of Nalidixicacid) for unknown bacterium

From the best fit line to the graph the y-axis interception (c1) and theslope (m1) are obtained:

c1 = −14.5 (mm)m1 = 26.3 (mm)

The formula for minimal inhibition concentration (MIC) is:

MIC1 = 10− c1

m1

The calculated value is:

MIC1 = 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 resultsare 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 2215 2010 185 15

2.5 90 —

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 Nalidixicacid) for E. coli

From the best fit line to the graph the y-axis interception (c2) and theslope (m2) are obtained:

c2 = 4.3 (mm)m2 = 13.7 (mm)

The formula for minimal inhibition concentration (MIC) is:

MIC2 = 10− c2

m2

The calculated value is:

MIC2 = 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- meansnegative control, C+ means positive control (E. coli), AK means the run ofPCR product of the unknown bacterium, KG and JK are the runs of PCRproducts of other bacteria and M means the run of commercially availableDNA standard marker. It is clearly seen that the amount of the producedDNA 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, H2S, TDA, IND, GLU, MAN, INO,SOR, RHA, SAC, MEL, AMY, ARA.

Sequencing results and comparison of the sequence dataof the unknown bacterium in website

The sequence of 16S rRNA of the unknown bacterium was compared in thewebsite (NCBI BLAST [10]). The hugest similarity was to Arthrobactersp., Micrococcus luteus and Micrococcus sp., Voriovorax sp., Antarctic bac-terium and Micrococcaceae bacterium among the 18 hits with highest scoreand longest nucleotide sequence for comparison. Arthrobacter sp. was inthe 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 andMicrococcaceae bacterium was in the 14th positions. All these hits had morethan 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. aregenuses 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 overthe yellowish ones because the dark yellow bacterium was more spread inthe Multivitamin juice at room temperature or just because they had slimycolonies which were prone to stick to the agar plate more than the yellowishones. The second possibility maybe the case because it was hard to take acolony of the dark yellow bacterium by a needle because it sticked better tothe agar than to the inoculation needle while the yellowish ones were easilytaken with the needle.

Gram staining of Eschericia coli and Bacillus subtilis

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

The shape of the colonies of the unknown bacteriumand 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 accordingto sequence comparison it is cocci but the shape of single bacterium underthe microscope was not spherical maybe because of the limited resolution ofthe microscope and distortions.

Gram staining of the unknown bacterium

Gram staining of the bacterium was incorrect because the Micrococcaceaefamily contains only gram-positive cocci. This is most probably becausethis sample was mixed with another one during gram staining. When thegram staining was performed three different bacteria were stained on thesame glass. Two of them were assigned incorrect gram staining results, onegram-positive, another gram-negative. And if these two results are changedto the opposite ones then the gram staining is in the accord with sequencecomparison 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 longerthan the theoretical one because the flask inoculated with E. coli was not allthe time in the warm incubator because the flask was taken out for severalminutes to take 0.5 ml sample for OD600 measurements and so the flask wascooled. Also one point in the graphs and data was disregarded because itintroduced a small peak.

Antibiotics resistance assays for E. coli, B. subtilis andthe unknown bacterium

B. subtilis is susceptable to all antibiotics with specific concentrations appliedat first time while E. coli and the unknown bacterium were not inhibited bythe initial concentration of the nalidixic acid. But during the second exper-iment the minimal inhibitory concentration of nalidixic acid was measuredand for E. coli it was MIC2 = 0.48 (mg/ml) and for the unknown bacteriumit was MIC1 = 3.6 (mg/ml). So the unknown bacterium is less susceptableto 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 becausenegative control (C-) was negative and the band of C+ and AK were at thesame 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, labmanual, 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 UnknownBacteria, lab manual, IUB[10] NCBI BLAST <http://www.ncbi.nlm.nih.gov/BLAST/> (visitedOctober 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> (visitedOctober 30, 2005)[12] Wikipedia, Micrococcaceae<http://en.wikipedia.org/wiki/Micrococcaceae> (visited October 30, 2005)

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