Characterization ofSerratia.spp. from Water and Sediment ...

Characterization of Serratia spp from Water and Sediment from Aquaculture Environment

Nurul Ain bt AbdulMattin

Bachelor of Science with Honours~~ pound6 (Resource Biotechnology) N974 2011 2011

PKHIDMAT MAKLUMAT AKADIMIK Pusat Khidmat Maklumat Aka emi~ Vpound t ~L vs P A

111111111 1111 1UUJLL

DECLARATION

I hereby declare that no portion of this dissertation has been submitted in support of an application for another degree ofqualification of this or any other university or institution of higher learning

I

Nurul Ain binti Abdul Mattin Resources Biotechnology Department of Molecular Biology Faculty of Resources Science and Technology University Malaysia Sarawak

II

ACKNOWLEDGEMENT

I am heartily thankful to my superVIsor Dr Samuel Lihan whose

encouragement guidance and support from the initial to the fmallevel of this whole

research project It is an honor for me to become one of his Final Year Projects

student and I am also very grateful for his willingness to always motivate me

towards completing my project and thesis write-up

I would also like to show my gratitude to my parents and beloved family

members who have given me a lot of spiritual and fmancial supports during this

whole project I would also want to thank Kathleen a master student in

Microbiology lab for willingly helping me throughout this project This research

project would not have been successful without her help

Lastly lowe my deepest gratitude to all my friends that have been helping

me around and motivating me whenever I feel like giving up Your kindness are

very much appreciated

I

III

PU3at Khidmat MakJumat Akadem ik UNIVERSITI MALAYSIA SARAWAK

TABLE OF CONTENT

Title amp Front Cover ~

Acknowledgement Table of contents III

List of Abbreviations IV

List ofTables and Figures ~ V

Abstracts 1

Chapter 1

Introduction 2

Chapter 2

Literature Review

20 Taxonomy bull 4

21 Morphology ampIdentification 4

22 Pathophysiology ofS marcescens 5

23 Epidemiology and Outbreaks Associated with Serratia spp 5

24 Identification ofSerratia spp by biochemical test 7

25 Characterization ofSerratia spp by Molecular Method 8 I

Chapter 3

Materials and Method

30 Sampling 10

31 Isolation and identification ofSerratia spp 10

32 Biochemical tests and DNA sequencing 11

15IV

I

shy

33 Characterization by molecular method

331 (GTG)5-PCR Analysis - 15

3321 Gel Electrophoresis 17

333 Antibiotic Susceptibility Testing 18

Chapter 4

Results amp Discussion

1940 Isolationof Serratia spp

41 Confirmation and Biochemical tests ofSerratia spp 21

42 Characterization of Serratia spp 30

420 (GT G)5-PCR Analysis 30


Conclusion 39

References 40

Appendices

i

v

LIST OF ABBREVIATIONS

AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic

Basic Local Alignment Search Tool

Cucurbit Yellow Vine Disease

Deoxyribonucleic acid

Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride

Polymerase Chain Reaction

Polyvinyl Chloride

Random Amplified Polymorphic DNA

Sulfide-Indo le-Motility

TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-

LIST OF TABLES AND FIGURES Page

Table 1 PCR reaction set-up for 16SrRNA 14

Table 2 PCR conditions for amplification of 16srRNA using 27fand 519r primers 15

Table 3 PCR reaction set-up for (GTG)s-PCR analysis 16

Table 4 PCR conditions for (GTG)s-PCR analysis 17

Table 5 Table of biochemical tests reaction 27

Table 6 Isolate codes and their species representatives 28

Table 7 Zone of inhibition (cm) of different types of antibiotics 35

Table 8 Antibiotic Resistance patterns and MAR (Multiple Antibiotic Resistance) Index 36

Table 9 Resistant Percentages () of the Types of Antibiotics Tested 37

Figure 1 Green-grey colonies of Serratia spp on PaIcam selective agar 19

Figure 2 Positive result ofTSI test 21

Figure 3 Positive result of catalase test 22

Figure 4 Positive result of SIM motility test 23

Figure 5 Gram negative with a cocci-shaped bacterium 24

Figure 6 (Left) Negative MR test (Right) Positive VP test 25

Figure 7 Agarose gel electrophoresis (15) of (GTG)s PCR of the Serratia spp 30

Figure 8 Dendogram of all isolates showing the degree of relatedness between each species typed 32

Figure 9 Antibiotic susceptibility testing of a sample 34

VII

Characterization ofSerratia spp from Water and Sediment Samples FromAquaculture Environment

Nurul Ain bt Abdul Mattin

Resource Biotechnology Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

Abstract

Serratia spp are classified under Enterobacteriaceae and the pathogenic species Serratia marcescens is notoriously

associated with nosocomial infection in patients In this study Serratia spp were isolated from water and sediment samples The sampling was carried out at a pond in Samariang and 7U1 Mile Kuching Further identification and

confmnation tests were carried out using TSI test citrate utilization test catalase test urease test MRVP test gram

staining and DNA sequencing (GTG)s-PCR was conducted to analyze the genetic diversity of species isolated II isolates of the Serratia spp were characterized by antibiotic susceptibility testing and DNA profiling using (GTG)sshy

PCR In antibiotic susceptibility testing the antibiotics used were ampicillin vancomycin bacitracin nitrofurantoin norfloxacin kanamycin erythromycin chloramphcnicol streptomycin tetracycline nalidixic acid sulphamethoxazoletrimethoprim gentamycin and carbenicillin All isolates were resistant to vancomycin bacitracin nitrofurantoin erythromycin and chloramphenicol The phylogenetic tree constructed for DNA fingerprinting using

(GTG)5-PCR technique indicates that Serratia spp isolated from the water and sediment samples were genetically

diverse and the isolates showed multiple-resistance to the antibiotics being tcsted

Key words Serratia spp biochemical tests DNA sequencing antibiotic susceptibility DNA fmgerprinting

Abstrak

Serratia spp dikategorikan di dalam kumpulan Enterobakter dan kewujudan strain pathogen Serratia marcescens biasanya bertanggungjawab di dalam jangkitan nosokomial di kalangan pesakit Di dalam kajian yang telah dilakukan ini spesis Serratia spp telah dipencilkan daripada persekitaran air dan mendapan tanah Persampelan telah dijalankan

di tasik berdekatanSwvariang dan Batu 7 Seterusnya pengesahan spesis tersebut telah dijalankan dengan menggunakan kaedah-kaedah biokimia seperti ujian TSI ujian pemanfaatan sitrat ujian katalase ujian urease ujian MRVP pewamaan gram dan teknik penjujukan DNA Kaedah penjujukan DNA dengan menggunakan (GTG)s-PCR berperanan untuk menguji diversiti genetik daripada pencilan yang telah dilakukan II pencilan Serratia spp juga telah

dicirikan menggunakan ujian ketahanan antibiotik dan pemprofilan DNA pula dijalankn menggunakan teknik (GTG)sshyPCR Antibiotik yang telah digunakan di dalam ujian ketahanan antibiotic adalah ampisilin vankomisin basitrasin nitrofurantoin norfloksasin kanamisin eritromisin k1oramfenikol streptomisin tetrasikl in asid nalidiksik

sulfametoksazoVtrimetoprim gentamisin dan karbenisilin Kesemua pencilan pula bertahan terhadap vankomisin basitasin nitrofurantoin eritromisin dan kloramfenikoL Pokok filogenetik yg telah dibuat daripada cap jari DNA menggunakan kaedah (GTG)s-PCR menunjukkan bahawa Serratia spp yang tclah dipencilkan daripada komponen air

dan mendapan mempunyai kepeJbagaian genetik yang luas dan pencilan tersebut menunjukkan kadar ketahanan antibiotik yang berbagai terhadap antibiotik yang diuji

Kata kunci Serralia spp ujian biokirnia penjujukan DNA ketahanan antibiotik cap jari DNA

1

CHAPTER

INTRODUCTION

Serratia spp are a Gram negative facultatively anaerobic and rod-shaped

bacteria Serratia spp is a genus under the family group of Enterobacteriaceae There are

6 species of Serratia spp namely S orodijera S rnbidaea S liquejaciens S

plymuthica S jonticola and the pathogenic strain of Serratia spp is known as S

marcescens S marcescens is associated with nosocomial infection in humans Infection

of the pathogenic strain of Serratia spp may cause some serious health complications

including meningitis and arthritis Serratia spp usually infect the bloodstream lower

respiratory and urinary tract Patients may experience fever chills and other serious

complications like endocarditis renal failure pneumonia chronic obstructive pulmonary

and urinary tract disease and many others

Serratia spp have become a considerable concern in causing chronic diseases due

to their wide distribution in the environments Based on a IS-month bacterial infection

surveillance a number of 732 babies were admitted to the neonatal care unit and S

marcescens had been reported to have been isolated in 153 babies (Smith et al 1984)

These alarming high rate of infection among neonatal had led to the research of Serratia

spp

Ongoing research on Serratia spp is still being conducted in order to know more

about how infectious it is to human beings and the best prevention and treatment method

Early identification of the presence of Serratia spp was done and several biochemical

tests was conducted to confirmed the presence of the bacteria from water and soil sample

2

Molecular identification of Serratia spp also was conducted using peR analysis and ~

antibiotic susceptibility testing was also conducted on the isolates DNA fingerprinting

was used to further characterize the Serratia spp strains isolated The significant of this

study was to detect the presence of Serratia spp that could indicate the presence of

pathogenic strain ofS marcescens

Hence the objectives of this study were

I) to isolate Serratia spp from water and sediment samples

2) to identify the isolated Serratia sppusing biochemical tests and DNA sequencing

3) to characterize the isolated strains using molecular DNA fingerprinting

3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy

The genus Serratia is categorized under the tribe Klebsiella and large family of

Enterobacteriaceae The 6 known species of Serratia are S orodifora S rubidaea S

Liquefaciens S plymuthica S fonticola and S marcescens S marcescens were first

discovered by a Venetian pharmacist in 1819 where he discovered a red discolouration

occurring in Polenta in Padua city (Sehdev amp Donnenberg 1998) Later in 1923 he

named the bacteria as Serratia in honor of an Italian physicist who invented steamboat

Serrafino Serrati The word marcescens in Latin means decaying Therefore the word

has been used together due to the rapid red color deterioration

21 Morphology and Identification

Serratia spp is a Gram negative rod-shaped bacterium It is a facultative anaerobe

and it is motie Serratia spp produces red pigments called prodiginines It is a highly

mucoid colonies when grown on agar Based on the 2nd edition of Bergeys manual of

Determinative Bacteriology Serratia spp falls under the family Enterobacteriaceae

lactose negative group It cannot ferment lactose and is indole-negative It gives a

negative result upon urease test and H2S production Serratia spp is a motile organism

4

II 1 hiJI IJN It

and citrate-positive It utilizes malonate and produces DNAse gelatinase lipase ~

lecithinase chitinase and esterase enzymes

22 Pathophysiology ofS marcescens

Infection by Serratia spp is responsible for the nosocomial infections of the

following sites bloodstream lower respiratory tract urinary tract surgical wounds and

epithelial tissues of adults The infection can also cause meningitis and arthritis in

pediatric ward patients Those addicted to heroin drug infection of the pathogenic strain

of S marcescens may lead to other serious complications including endocarditis and

osteomyelitis

Infection of S marcescens reported may also cause 2 types of pneumonia in the

year of 1968-1980 nine patients experienced acute haemorrhagic bronchopneumonia

and seven more had diffuse neutropenic vasculitis (Goldstein et ai 1982)

23 Epidemiology and Outbreaks Associated with Serratia spp

In October 1999 7 patients had been reported to have been infected with S

marcescens in a hospital located in Ontario Canada Among them 5 patients suffered ~

bull r

from bacteremia while 2 of them suffered from wound infections Among 5 of them who

showed presence ofbacteria in their blood 2 of them died later (Bonnie et ai 2001)

There was also an outbreak occurring from September 1998 to June 1999 which

involved 24 patients admitted in the bone marrow transplant and oncology unit 14 of

them developed very serious infection due to the presence of Serratia marcescens in their

5

I

blood Serratia spp were isolated from several sources including the control buttons of

intravenous infusion pump and urine jug assigned to a patient without evidence of S

marcescens carriage (Knowles et at 2000)

In Zurich (April 1998-May 1999) S marcescens pathogen had been isolated from

bottles of liquid theophylline Eleven out of 20 neonated had been infected and the

colonization of pathogen occurred within 24 hours after delivery Isolates were obtained

from stool and gastric aspirate specimens (Fleisch et at 2002)

In 2000 an outbreak in a number of Scotland NICUs (neonatal intensive care

units) had been reported This outbreak occurred over 6 week period where 12 babies

were being admitted at the Glasgow Royal Maternity Hospital (GRMH) and 5 more

babies at the Queen Mothers Hospital (QMH) Among those admitted in GRMH 3

babies suffered from septicaemia and 2 of them actually died After subsequent

investigations the outbreak pathogenic strain of S marcescens had been proved to have

been successfully isolated from laryngoscope blade and an expressed breast milk sample

(Jones et at 2000)

In 2006 at the Farabi Hospital of Karadeniz Technical University in Trabzon

Turkey 3 of 9 neonates admitted were dead due to the presence ofS marcescens in their

blood After investigation the pathogenic bacteria were found in one hand-washing

sample and two breast milk samples (Bayramoqluet at 2011)

6

24 Identification of Serratia spp by biochemical test

Serratia spp is a motile organism and can grow in temperatures ranging from 5shy

40degC and pH levels ranging from 5 to 9 It can be distinguished by other types of

Enterobacteriaceae by its ability to hydrolyze casein which allows it to produce

extracellular metaUoproteinases S marcescens is able to degrade tryptophan and citrate

Degradation of tryptophan may yield pyruvic acid as by-product which later be used by

S marcescens in their metabolic processes Degradation of citrate on the other hand will

yield carbon which is used as a C source

Methyl red test is used to determine whether the bacteria is undergoing mixed-

acid fermentation or not In the case of S marcescens the result of methyl red test is

supposed to be negative Other than that S marcescens is able to ferment lactose

producing lactic acid as the end-product TSI reaction were acid-butt with no production

of hydrogen sulfide The bacteria is motile and did not give positive result for indole test

MR test were negative and the opposite positive result in VP test

In spirit blue lipase test S marcescens give positive result in which the presence

of clearing zone surrounding the sample This differential test determines whether the

organism produ~es the secreted lipase enzymes S marcescens also are able to digest

gelatin Indication of positive result is shown by the liquefaction of the media upon

refrigeration

S marcescens gives positive result upon catalase test in which visible bubbles

appear upon contact with hydrogen peroxide Negative result is obtained with oxidase

test

7

shy

25 Characterization ofSerratia spp by Molecular Method

Based on article by Kur et al (1995) molecular methods that had been used in the

epidemiological study of Serratia spp includes the conventional PCR This method had

successfully detected 40 clinical strains of the pathogenic strain of S marcescens using a

primer that amplifY the spacer regions between the 16S and 23S genes in the prokaryotic

rDNA loci A combination of biotyping and RAPD-PCR method had also been

conducted in the isolates from the nasocomial infection of pediatric patients This method

shows the clonal variations available in the sample isolated (Enciso-Moreno et ai 2004)

A genotyping study using rep-PCR of the strain S marcescens that causes CYVD

(cucurbit yellow vine disease) showed the banding pattern were identical to the strains

genotyped using the DNA-DNA hybridization technique (Zhang et aI 2003) In other

study rep-pcr using the primer (GTG)s also had been done in genotyping analysis in 2

premature infants that suffered from the S marcescens infection and the result showed

that 3 out of 5 sample of patients involved were genetically-related ( Campbell et ai

1998)

In Spain 38 isolates from different oak species that has been reported as a J

I

causative agents of causing cankers in oak trees from different locations was analyzed by

sequencing using 16SrDNA and rep-PCR fingerprinting method The sequencing result

showed that 34 out of 38 isolates were from Brenneria spp and 4 of the isolates were

from Serratia spp Dendogram obtained showed that Serratia spp also are

phylogenetic ally close to the Brenneria spp (Gallego et ai 2008)

8

A different study carried out in Korea proved that RAPD-PCR ERIC-PCR and ~

Rep-PCR were a reliable method in genotyping studies of the strain S marcescens All

banding patterns from the 24 samples obtained from patients admitted at the Changbuk

National University Hospital were identical in the same epidemic strain and unidentical

in non-epidemic strain ( Shin 2003)

9

CHAPTER 3

MATERIALS AND METHOD

30 Sampling

Water (50ml) and soil (25g) samples were taken from Samariang and 7th Mile

aquaculture pond The sediments were collected using a PVC pipe These samples were

transported to the laboratory in ice container Samples brought to the lab were then

immediately processed pH of the water samples were obtained and recorded using a pH

meter

31 Isolation and identification ofSerratia spp

310 Isolation using Palcam Selective agar

225ml of Fraser broth were used for enrichment An equal amount of samples

from 3 random area for each water and soil samples were added into the broth and mixed

1 ml of sample from Fraser broth was then added into a 9ml of saline-containing tubes

(100 ml distilled water in 085g NaCl) yielding a total of 10ml solution Serial dilution of

101 102 103 and 104 were prepared using the standard technique

1 00 ~l of each dilution tube was then spread out onto Palcam selective agar

Then the plate were incubated for 24 hours at 29degC After incubation each colony were

10

first being streaked and incubated onto non-selective slant TSA agar to get pure culture ~

for biochemical confirmation tests

32 Biochemical tests

320 TSI test

The agar was first prepared Then a colony of the bacterial isolate was then

stabbed into the center of the tubes containing the medium agar using a sterile needle

After that the needle was streaked back and forth onto the surface of the slanted medium

and be incubated for not more than 24 hours The TSI agar contains three sugars

(dextrose lactose and sucrose) phenol red to detect carbohydrate fermentation and

ferrous ammonium sulphate for detection of hydrogen sulfite production The caps were

closed loosely upon incubation to permit free exchange of air so as to enhance the

alkaline condition of the slant

321 Citrate utilization test

After prepanng the Simmons citrate medium bacterial colonies from the

cultured slant agar were picked by a sterile wire loop and streaked onto the medium The

streaked plate were then incubated overnight 322 Catalase test

Bacterial colony were cultured onto a non-selective TSA agar and incubated for

24 hours After cultivation 2 or 3 drops of catalase (hydrogen peroxide) reagent were

added onto the culture in the plate Formation of bubbles upon addition were observed

and record ed

11

323 Urease test

After preparing the urease agar the isolates were streaked onto the medium by a

wire loop The plate were incubated for 24 hours and the color changes of the medium

were observed

324 81M test

Motility test was carried out in this study to determine whether isolated bacteria

was motile or not Medium used was the BBLTM 81M motility test medium A colony

from the pure culture was stabbed through the center of the jelly-like consistency of the

medium The tubes containing the cultured medium were incubated for 24 hours After

incubation the appearance of the agar were observed and analyzed

325 MRVP test

The isolates were first cultured into sterile LB broth After cultivation the culture

was divided into two sterile bottles to perform the MR (methyl red) and VP (vogesshy

proskauer) test

F or methyl red (MR) test 2 or 3 drops of methyl red reagent were added into the

bottle and cdlorlchanges were observed

Meanwhile voges-proskauer (VP) test were conducted using Barrits reagent A

and B A few drops of each reagent were added subsequently and the color changes were

observed

12

326 Gram Staining

This test detennined whether the bacterium is either Gram positive or Gram

negative indicated by the colour of the stain obsetved under the microscope

First the cultured isolates were streaked and fixed onto a clean glass slide using a

Bunsen burner flame The smear was then drained with a few drops of the primary stain

crystal violet for 1 minute This primary stain renders all the bacteria unifonnly into

violet colour Then the excess stain were washed out under rwming tap water The smear

were then treated with a few drops of a mordant iodine for 1 minute The slides were

then washed under running water before being decolourized in alcohol for only about a

few seconds Prolonged exposure to alcohol may cause over-decolorization Then the

slides were washed under rwming tap water before treating them with the red

counterstain safranin for 1 minute Tills stain gives the colour red in Gram staining

Excess safranin were then washed out and the slide were let to air-dried before

observation of the bacterial morphology under the microscope

327 DNA Sequencing

I 01

DNA sequencing was carried out by amplifying 16SrRNA of the bacterial isolates

using universal primers 27f and 519r (Hutter et al 2003) The primers sequence were

27f (5-AGAGTTTGATCMTGGCTCAG-3)

519r (5-GWATTACCGCGGCKGCTG-3)

13

The PCR reaction set-up used consists of the following (Table 1)

Table 1 PCR reaction set-up for 16SrRNA

peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~

P CR conditions for 16srRNA amplification of bacterial isolates were as follows

(Table 2)

Table 2 PCR co nditions for amplification of 16srRNA using 27fand 519r primers

Steps

Initial Denaturati on

Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec

Annealing 55degC amp 58 degc respectively 1 min

Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s

T he PCR products were then purified using Qiagen DNA purification kit The

pure DN A (elution of 200 d) was sent to First Base Sdn Bhd for DNA sequencing

services

33 Characterization by Molecular Method

331 (GTG)s-PCR Analysis

The (GTG)s-PCR was carried out according to the method described by Matsheka

et al (2006) The primer used was (GTG)s The sequence ofthe primer is shown below

(GTG)s 5-GTG GTG GTG GTG GTG-3

15


Table 3 PCR reaction set-up of (GTG)5-PCR

peR components Volume

Taq Polymerase (2U) 03 III

(GTG)5 primer 1III

MgCh 3111

dNTP 081l1

Distilled H2O 99 III

Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16

PCR conditions for (GTG)s-PCR amplification of bacterial isolates were as follows

(Table 4)

Table 4 PCR conditions for (GTG)s-PCR analysis

Steps I Temperature Time I I

Initial Denaturation 5 min 93degC

Denaturation 95degC 30 sec

Annealing 70degC 1 min

Extension 65degC 8 min

Final Extension 65degC 16 min

3 o

c y c

e s

3321 Gel Electrophoresis

Electrophoresis gel was prepared first by melting 075 g of agarose powder in 50

ml of TBE to obtain 15 of agarose concentration Then the gel was let cooled and

poured into a casting tray Four microlitres of samples were loaded into the well together ~

with DNA ladder~~d negative control for comparison The DNA ladder were mixed with

loading dye for ease of analysis Then electrophoresis were run on an electrode chamber

starting at 100 miliamperes (rnA) for 1 hour and 30 minutes Then the gel mold were

stained in Ethidium Bromide (EtBr) for 20 minutes Destaining of the gel mold took

about 15 minutes The staining and de staining process were conducted in the dark room

17

PKHIDMAT MAKLUMAT AKADIMIK Pusat Khidmat Maklumat Aka emi~ Vpound t ~L vs P A

111111111 1111 1UUJLL

DECLARATION

I hereby declare that no portion of this dissertation has been submitted in support of an application for another degree ofqualification of this or any other university or institution of higher learning

I

Nurul Ain binti Abdul Mattin Resources Biotechnology Department of Molecular Biology Faculty of Resources Science and Technology University Malaysia Sarawak

II

ACKNOWLEDGEMENT














I

III


TABLE OF CONTENT





Abstracts 1

Chapter 1

Introduction 2

Chapter 2

Literature Review

20 Taxonomy bull 4






Chapter 3


30 Sampling 10



15IV

I

shy





Chapter 4







Conclusion 39

References 40

Appendices

i

v


AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic




Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride


Polyvinyl Chloride



TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

ACKNOWLEDGEMENT














I

III


TABLE OF CONTENT





Abstracts 1

Chapter 1

Introduction 2

Chapter 2

Literature Review

20 Taxonomy bull 4






Chapter 3


30 Sampling 10



15IV

I

shy





Chapter 4







Conclusion 39

References 40

Appendices

i

v


AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic




Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride


Polyvinyl Chloride



TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17


TABLE OF CONTENT





Abstracts 1

Chapter 1

Introduction 2

Chapter 2

Literature Review

20 Taxonomy bull 4






Chapter 3


30 Sampling 10



15IV

I

shy





Chapter 4







Conclusion 39

References 40

Appendices

i

v


AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic




Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride


Polyvinyl Chloride



TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

I

shy





Chapter 4







Conclusion 39

References 40

Appendices

i

v


AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic




Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride


Polyvinyl Chloride



TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17


AB

BLAST

CYVD

DNA

EtBr

HCL

LB

MHA

MR

NaCI

PCR

PVC

RAPD

SIM

TBE

TSA

TSI

VP

Antibiotic




Ethidium Bromide

Hydrochloric acid

Luria broth

Mueller-Hinton agar

Methyl red

So dim chloride


Polyvinyl Chloride



TrislBoratelEDT A

Trypticase soy agar

r Triple sugar iron

Voges-Proskauer

VI

-




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17




















VII






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17






Abstract









Abstrak







1

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

CHAPTER

INTRODUCTION
















spp





2










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17










3

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

CHAPTER 2

LITERATURE REVIEW

20 Taxonomy
















4

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

II 1 hiJI IJN It









osteomyelitis







bull r






5

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

I















(Jones et at 2000)





6



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17



















refrigeration



test

7

shy















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17















1998)


I






8






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17






9

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

CHAPTER 3


30 Sampling





meter










10




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17




320 TSI test
















and record ed

11

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

323 Urease test



were observed

324 81M test






325 MRVP test



proskauer) test





observed

12

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

326 Gram Staining














327 DNA Sequencing

I 01





13



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17



peR components

Taq Polymerase (2U)

27fand 519rprimers

MgCh

dNTPmix

Distilled H2O

Buffer (1 Ox)

Template DNA

Total

Vohime

04 )11

1)11

3 )11

1)11

46 )11

5 )11

10 )11

25 III

14

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17

- I~


(Table 2)


Steps


Denaturation

Temperature

95degC

94 degc

Time

10 min

30 sec


Extension

Final Extension

72degC

72degC

1 min 30 sec

20 min

2 6

c y c I e s



services






15





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17





(GTG)5 primer 1III

MgCh 3111

dNTP 081l1


Buffer (lax)

5 III

Template DNA 5 III

Total

I

25 III

16


(Table 4)








3 o

c y c

e s










17


(Table 4)








3 o

c y c

e s










17

Characterization ofSerratia.spp. from Water and Sediment ...

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