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  • i

    Early Larval Development and Identification of Infectious

    Microbes and Antibiotic Resistance in Hybrid Climbing

    Perch, Anabas sp.

    by

    Normasarah bt Hj Ab Rahman

    A thesis submitted in fulfillment of the requirements for the degree of

    Master of Science

    Faculty of Agro Based Industry

    UNIVERSITI MALAYASIA KELANTAN

    2016

  • ii

    Early Larval Development and Identification of

    Infectious Microbes and Antibiotic Resistance in

    Hybrid Climbing Perch, Anabas sp.

    NORMASARAH BT HJ AB RAHMAN

    MASTER OF SCIENCE

    2016

  • iii

    THESIS DECLARATION

    I hereby certify that the work embodied in this thesis is the result of the original

    research and has not been submitted for a higher degree of any other University or

    Institution.

    OPEN ACCESS I agree that my thesis is to be made immediately

    available as hardcopy or on-line open access (full text)

    EMBARGOES I agree that my thesis is to be made available as hardcopy

    or on-line (full text) for a period approved by the Post

    Graduate Committee.

    Dated from until .

    CONFIDENTIAL (Contains confidential information under the Official

    Secret Act 1972)*

    RESTRICTED (Contains restricted information as specified by the

    organization where research was done)*

    I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

    1. The thesis is the property of Universiti Malaysia Kelantan.

    2. The library of Universiti Malaysia Kelantan has the right to make copies for the

    purpose of research only.

    3. The library has the right to make copies of the thesis for academic exchange.

    SIGNATURE SIGNATURE OF SUPERVISOR

    IC/ PASSPORT NO. DR LEE SEONG WEI

    Date: Date:

  • iv

    ACKNOWLEDGEMENT

    Alhamdulillah, praise to Allah S.W.T, for His blessing and mercy that enabled

    me complete this thesis.

    Firstly, I would like to express my deepest gratitude and appreciation to my

    supervisor, Dr. Lee Seong Wei for his guidance, constructive comments,

    encouragement and support throughout the study on „Early larval development and

    identification of infectious microbes and antibiotic resistance hybrid climbing perch,

    Anabas sp.‟

    Special thanks to Mr. Khalid for guidance in the laboratory and help throughout

    this study. My appreciation to my friends especially all post graduate students for their

    support. My deepest gratitude to my colleagues, Ms. Husna, Ms. Afiqah, Ms. Suaini,

    Ms. Farisya and Mrs. Azwahanis for their support throughout this study. I am also

    grateful to all my family members, especially my parents Haji Abdul Rahman Hamat

    and Hajjah Che Hamidah Hassan, my uncle Adnan Hassan and my husband Asrul

    Afandi Halim for their endless prayers and moral support.

    Finally, I would like to thank Universiti Malaysia Kelantan especially Faculty

    of Agro Based Industry for the research facilities and MyBrain for the scholarship

    provided to me. My special thanks goes to Ministry of Higher Education, Malaysia

    under Skill Niche Research Grant scheme (NRGS)

    (R/NRGS/A07.00/00387A/006/2014/000152) for funding this research activity. Last

    but not least, it is my pleasure to thank all others who have contributed either directly

    or indirectly give their support and guidance.

  • v

    TABLE OF CONTENTS

    PAGE

    THESIS DECLARATION i

    ACKNOWLEDGEMENT ii

    TABLE OF CONTENTS Iii

    LIST OF TABLES Viii

    LIST OF FIGURES X

    LIST OF ABBREVIATIONS Xiii

    LIST OF SYMBOLS Xiv

    ABSTRACT Xv

    ABSTRAK Xvi

    CHAPTER 1 INTRODUCTION

    1.1 Background of the Study 1

    1.2 Statement of the Problem 2

    1.3 Objectives of the Study 3

    1.4 Scope of the Study 4

    1.5 Significances of the Study 4

    1.6 Organisation of Thesis 5

    CHAPTER 2 LITERATURE REVIEW

    2.1 Introduction 6

    2.2 General Characteristic and Taxonomy of Anabas testudineus 6

    2.3 Early Development of Anabas testudineus 10

    2.4 Water Parameter on Fish Larvae Development 12

    2.4.1 Effect of Temperature on Early Development of Fish Larvae 12

    2.4.2 Effect of Salinity on Early Development of Fish Larvae 14

    2.4.3 Effect of pH on Early Development of Fish Larvae 16

    2.4.4 Effect of Formalin on Early Development of Fish Larvae 18

  • vi

    2.5 Bacterial Pathogens and Disease of Aquatic Organisms 19

    2.6 Isolation and Identification of Bacterial from Aquaculture Sides 23

    2.7 Characterization of Antibiogram for Salmonella sp. and Aeromonas sp. 24

    2.8 Summary 29

    CHAPTER 3 MATERIALS AND METHODS

    3.1 Introduction 31

    3.2 Material and Preparation of Sample 31

    3.3 Process of Breeding Broodstock of Hybrid Anabas Species 32

    3.4 Experiments on Environmental Effects During Early Development of

    Hybrid Anabas Species

    35

    3.4.1 Effect of Temperature 36

    3.4.2 Effect of pH 37

    3.4.3 Effect of Salinity 38

    3.4.4 Effect of Formalin 39

    3.5 Method to Isolate and Identify Type of Bacteria on Infected Hybrid

    Anabas species

    39

    3.5.1 Sampling and Isolation of Bacteria 40

    3.5.2 Tests to Identify Type of Bacteria on Infected Hybrid Anabas 41

    3.6

    species

    Methods to Characterize Antibiogram of Bacteria Isolated for Infected

    43

    Hybrid Anabas Species

    3.6.1 Blood Haemolysis Test

    43

    3.6.2 Antibiotic Susceptibility Test 44

    3.7 Statistical Analysis 45

    3.8 Summary 46

  • vii

    CHAPTER 4 RESULTS

    4.1 Introduction 47

    4.2 Fertilization and Hatching 47

    4.3 Phases on Early Development of Hybrid Anabas Species 48

    4.4 Results on Environmental Effects on Early Development of Hybrid

    Anabas species

    58

    4.4.1 Effect of Temperature 59

    4.4.2 Effect of pH 61

    4.4.3 Effect of Salinity Concentration 62

    4.4.4 Effect of Formalin Concentration 64

    4.5 Isolation of Bacteria 66

    4.6 Bacterial Identification 67

    4.7 Blood Haemolysis Test 71

    4.8 Antibiotics Susceptibility Test 72

    4.8.1 Multiple Antibiotic Resistances (MAR) 75

    4.9 Summary 75

    CHAPTER 5 DISCUSSION

    5.1 Introduction 77

    5.2 Early Development of Hybrid Anabas species 77

    5.2.1 Effect of Temperature 79

    5.2.2 Effect of pH 81

    5.2.3 Effect of Salinity Concentration 83

    5.2.4 Effect of Formalin Concentration 85

    5.3 Isolation and identification of Bacteria 88

    5.4 Identification of Bacteria Salmonella sp. and Aeromonas sp. 89

    5.5 Blood Haemolysis Test 90

  • viii

    5.5 Antibiotic Susceptibility Test 91

    5.5.1 Multiple Antibiotic Resistance (MAR) 96

    5.7 Limitation of the study 97

    5.8 Implication of the Study 98

    5.9 Summary 98

    CHAPTER 6 CONCLUSION AND FUTURE WORKS

    6.1 Conclusions 99

    6.2 Suggestions for Future Works 101

    REFERENCES 102

    APPENDICES 111

  • ix

    LIST OF TABLES

    NO. PAGE

    4.1 Embryonic development stage of hybrid Anabas species 48

    4.2 Larvae development stage of hybrid Anabas species 55

    4.3 Number of suspected bacteria based on different isolation bacteria 66

    media

    4.4 Phenotypic characteristics of bacterial isolates, Salmonella sp. 68

    4.5 Phenotypic characteristics of bacterial isolates, Aeromonas sp. 70

  • x

    LIST OF FIGURES

    NO. PAGE

    2.1 Thailand Anabas testudineus 5

    2.2 Local Anabas testudineus 6

    3.1 Process of breeding broodstock 34

    4.1 Total times of hatching period, egg yolk absorption period and 59

    starvation period emphasis on pH parameter

    4.2 Total times of hatching period, egg yolk absorption period and 61

    starvation period emphasis on temperature parameter

    4.3 Total times of hatching period, egg yolk absorption period and 63

    starvation period emphasis on salinity parameter

    4.4 Total times of hatching period, egg yolk absorption period and 65

    starvation period emphasis on formalin parameter

    4.5 Total percentage (%) of antibiogram on isolate bacteria, 73

    Salmonella sp.

    4.6 Total percentage (%) of antibiogram on isolate bacteria, 74

    Aeromonas sp.

  • xi

    LIST OF ABBREVIATIONS

    AML Amoxycilin

    AMP Ampicilin

    C Chloramphenicol

    CT Colistin Sulphate

    DO Doxycycline

    E Erythromycin

    F Florfenicol

    FOS Fosfomycin

    H(s) Hour(s)

    Min(s) Minute(s)

    MAR Multiple antibiotic resistance

    MY Lincomycin

    NA Nalidixic acid

    NV Novobicin

    OA Oxolinic acid

    OL Oleondomycin

    S3 Sulphonimides

    SP Spiramycin

    TE Tetracycline

    TSA Tryptic soy agar

    TSB Tryptic soy broth

    GSP Glut

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