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Study of Population Dynamics of Cockroaches Collected from Lahore with Resistance Patterns of Their Isolated Microbial Fauna” Submitted by Ms

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Study of Population Dynamics of Cockroaches Collected from Lahore with Resistance Patterns of Their Isolated Microbial Fauna” Submitted by Ms STUDY OF POPULATION DYNAMICS OF COCKROACHES COLLECTED FROM LAHORE WITH RESISTANCE PATTERNS OF THEIR ISOLATED MICROBIAL FAUNA _____________________________________________________________________ HAFSA MEMONA ________________________________________________________________________ DEPARTMENT OF ZOOLOGY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2016 STUDY OF POPULATION DYNAMICS OF COCKROACHES COLLECTED FROM LAHORE WITH RESISTANCE PATTERNS OF THEIR ISOLATED MICROBIAL FAUNA _____________________________________________________________________ A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY By HAFSA MEMONA ________________________________________________________________________ DEPARTMENT OF ZOOLOGY LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2016 CERTIFICATE This is to certify that the research work described in thesis entitled “Study of population dynamics of cockroaches collected from lahore with resistance patterns of their isolated microbial fauna” submitted by Ms. HAFSA MEMONA to Department of ZOOLOGY, Lahore College for Women University has been carried out under our direct supervision. We have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. We further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. We also certify that the enclosed manuscript has been prepared under my supervision and we endorse its evaluation for the award of PhD degree through the official procedure of University. ________________ _____________________ Supervisor Co-supervisor Prof. Dr. Farkhanda Manzoor Prof. Dr. Aftab Ahmad Anjum Head Department of Zoology Chairman Department of Microbiology LCWU, Lahore. UVAS, Lahore. Date: Verified By Chairman ________________ Prof. Dr. Farkhanda Manzoor Head Department of Zoology Stamp: _________________ Controller of Examination Stamp Date: ___________ DEDICATION I DEDICATE ALL MY SUCCESS TO MY EVER LOVING PARENTS, PROF. AHMAD YAR KHAN AND MRS. AZRA PARVEEN AND MY KIND HEARTED GRANDMOTHER, MRS. SARDAR BEGHUM (LATE) WHOSE INSPIRATION, ENCOURAGEMENT, SACRIFICE, MORAL ENTHUSIASM, FINANCIAL SUPPORT, GUIDANCE, EXPERIENCE, WISDOM AND KNOWLEDGE MADE THIS STUDY POSSIBLE. OTHERWISE, WITHOUT THEIR HELP IT WOULD HAVE BEEN A DREAM FOR ME ACKNOWLEDGMENTS The whole praise to ALLAH Almighty the sovereign Power, the creator of the universe, who gave us knowledge, with which we are trying to understand the laws of nature and conquer the universe; who made me the super creature; blessed me with knowledge and enabled me to accomplish this task. I offer my humblest and sincere words of thanks to the Holy Prophet Muhammad (PBUH), who is forever a torch of guidance for humanity. A special acknowledgement goes to Higher Education Commission, Pakistan for financial support in this fully funded research by awarding me Indigenous scholarship. I feel all words on the earth just failing to express my deep sense of gratitude to our respected Vice Chancelor, Prof. Dr. Uzma Qureshi for giving me opportunities to complete my research thesis of Ph.D Zoology. I respectfully express my deepest appreciation to our respected, distinguished and affectionate director research Prof. Dr. Shagufta Naz for providing opportunities to complete my research thesis. I am also obliged to my supervisor Prof. Dr. Farkhanda Manzooor, Head of Zoology Department for her sincerest guidance, critical suggestions. She has been so kind and supportive with her patience and knowledge and a source of inspiration for me. I respectfully express my deepest appreciation and acknowledgement to my co-supervisor Prof. Dr. Aftab Ahmad Anjum, Chairman of Microbiology Department, UVAS, Lahore for his keen interest, providing research facilities and valuable suggestions. I am also greatly indebted to my friends, lab fellows for their friendly attitude, support and sympathetic encouragements. Special thanks to the incharge, Urban Pest Management (UPM) and their team for helping me in sample collection. I also want to pay thanks to laboratory staff of Zoology Department of LCWU for their cooperation. HAFSA MEMONA CONTENTS Title Page no. List of Tables i List of Figures iii List of Abbreviations vi Abstract xi Chapter 1 : Introduction 1 Chapter 2: Literature Review 15 Chapter 3: Materials and Methods 44 3.1: Experimental sites 44 3.2: Study area 44 3.3: Collection and identification of cockroaches 44 3.3.1: Collection of cockroaches 44 3.3.2: Identification of cockroaches 47 3.3.3: Weather data collection 47 3.3.4: Data analysis 47 3.4: Isolation and identification of microbes from external surfaces 49 of cockroaches 3.4.1: Sample collection 49 3.4.2: Isolation of bacteria from external surfaces of 49 cockroaches 3.4.3: Identification of bacteria from external surfaces of 49 cockroaches 3.4.3.1: Preparation of media 49 3.4.3.1.1: Preparation of Tryptic Soy Agar 49 (TSA) 3.4.3.1.2: Preparation of Mannitol Salt Agar 49 (MSA) 3.4.3.1.3: Preparation of MacConkey Agar 50 3.4.3.1.4: Preparation of Eosin Methylene 50 Blue Agar (EMB) 3.4.3.1.5: Preparation of Salmonella shigella 50 Agar (SS agar) 3.4.3.1.6: Preparation of Blood Agar Plate 50 (BAP) 3.4.4: Inoculation of sample 51 3.4.5: Identification of microbes 51 3.4.5.1: Microscopic colonial morphology 51 3.4.5.2: Gram staining 51 3.4.5.3: Biochemical tests 52 3.4.5.3.1: Oxidase test 52 3.4.5.3.2: Catalase test 52 3.4.5.3.3: Coagulase test 53 3.4.5.3.3.1: Slide coagulase test 53 3.4.5.3.3.2: Tube coagulase test 53 3.4.5.3.3: Indole test 53 3.4.5.3.4: Methyle Red -Voges Proskaeur 54 (MRVP) test 3.4.5.3.4.1: Methyl red test 54 3.4.5.3.4.2: Voges-Proskauer (VP) 54 test 3.4.5.3.5: Citrate test 55 3.4.6: Statistical analysis of bacterial isolates 55 3.5: Isolation and identification of bacteria from alimentary tract of 57 cockroaches 3.5.1: Collection of samples 57 3.5.2: Isolation of bacteria from alimentary tract of 57 cockroaches 3.5.3: Identification of microbes from alimentary tract of 57 cockroaches 3.5.3.1: Preparation of media 57 3.5.3.2: Inoculation of sample 57 3.5.3.3: Identification of microbes 57 3.5.4: Statistical analysis of microbial isolates from 58 alimentary tract of cockroaches 3.6: Isolation of fungal flora from cockroaches 59 3.6.1: Preparation of samples 59 3.6.2: Preparation of media for fungal culturing 59 3.6.2.1: Preparation of Sabouraud’s Dextrose Agar 59 (SDA) 3.6.2.2: Preparation of Malt Extract Agar (MEA) 60 3.6.3: Inoculation of samples 60 3.6.4: Identification of fungal isolates 61 3.7: Isolation of parasitic contaminants from external surfaces of 61 cockroaches 3.7.1: Preparation of samples 61 3.7.2: Identification of parasitic contaminants 61 3.7.3: Statistical analysis 62 3.8: Evaluation of antimicrobial sensitivity 62 3.8.1: Preparation of Tryptic Soy Broth (TSB) 63 3.8.2: Inoculation of sample in Tryptic Soy Broth (TSB) 63 3.8.3: Preparation of Mueller Hinton Agar plates 63 3.8.4: Inoculation of samples on Muller Hinton Agar 64 3.8.5: Analysis of results 64 3.9: Efficacy of common disinfectants for bacterial isolates 64 3.9.1: Preparation of MH Agar plates 65 3.9.2: Inoculation of samples 65 3.9.3: Serial dilution of disinfectants 65 3.9.4: Well diffusion method 65 3.10: Quantitative and qualitative analysis of total protein extracted 66 from resistant bacterial isolates 3.10.1: Preparation of reagents 67 3.10.1.1: 1X Phosphate Buffer Saline (PBS) 67 3.10.1.2: 1X cell lysis buffer 67 3.10.1.3: Phenylmethylsulfonyl fluoride (PMSF) 67 solution 3.10.2: Protein extraction procedure 67 3.10.3: Bradford protein assay 67 3.10.3.1: Preparation of Bradford reagent 68 3.10.3.2: Preparation of dilutions of standard protein 68 sample {Bovine Serum Albumin (BSA)} 3.10.3.3: Macro assay 68 3.10.3.4: Calculations for unknown samples 68 3.10.4: SDS-Polyacrylamide Gel Electrophoresis (SDS- 69 PAGE) 3.10.4.1: Reagent preparation 69 3.10.4.1.1: 30% Acrylamide/ Bisacrylamide 69 solution 3.10.4.1.2: Tris-HCl 1.5 M (pH 8.8) 69 Resolving gel buffer 3.10.4.1.3: 1 M Tris-HCl (pH 6.8) Stacking 69 gel buffer 3.10.4.1.4: 10% Sodium Dodecyl Sulphate 69 (SDS) Solution 3.10.4.1.5: 10% Ammonium per Sulphate 70 (APS) solution 3.10.4.1.6: 1X Running buffer (SDS 70 electrophoresis buffer) 3.10.4.1.7: Tracking dye (loading dye) 70 3.10.4.1.8: Coomassie stain (staining 70 solution) 3.10.4.1.9: Coomassie destain (destaining 70 solution) 3.10.4.1.10: Preparation of working dilutions 70 3.10.4.1.11: Protein marker 71 3.10.4.2: Gel preparation 71 3.10.4.2.1: 10% Resolving gel preparation 71 3.10.4.2.2: 5% Stacking gel preparation 71 3.10.4.3: Gel electrophoresis 71 3.10.4.4: Staining 72 3.10.4.5: Destaining 72 3.10.4.6: Image capture and photography 72 3.10.4.7: Quantification of protein fractions 72 Chapter 4: Results 73 4.1: Collection and Identification of Cockroaches 73 4.1.1: Identified species of cockroaches 73 4.1.2: Distribution and abundance of cockroach species 76 4.1.2.1: First Trimester 76 4.1.2.2: Second Trimester 76 4.1.2.3: Third Trimester 77 4.1.2.4: Fourth Trimester 77 4.1.2.5: Relative abundance 79 4.2: Isolation of microorganisms from external and internal surfaces 83 of cockroaches 4.2.1: Identified bacterial species 83 4.2.2: Bacterial infection rate at hospitals and houses 88 4.2.3: Ecological indices of isolated microorganisms 90 4.3: Isolation of fungal flora from cockroaches 91 4.3.1: Variety of fungal isolates 91 4.3.2: Prevalence of fungal flora isolated from P.
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