ANTIMICROBIAL ACTIVITY AND QUALITATIVE PHYTOCHEMICAL COMPOSITION OF CRUDE EXTRACTS FROM MEDICINAL PLANTS AGAINST SELECTED ENTERIC BACTERIAL PATHOGENS AND Candida albicans HIBERT RACHUONYO OPINDE (BSc) I56/20520/2012 A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Microbiology) in the School of Pure and Applied Sciences of Kenyatta University September 2016 i DECLARATION This thesis is my original work and has not been presented for a degree in any other University. Signature ………..……………… Date ……..…….…… Hibert Rachuonyo Opinde Department of Microbiology Kenyatta University This thesis has been submitted for examination with our approval as the University supervisors. Signature ………………………... Date.…….....…….....…. Dr. Anthony Kebira Department of Microbiology Kenyatta University Signature ……………………….. Date.……......……...….. Dr. Grace W. Gatheri Department of Plant Sciences Kenyatta University ii DEDICATION This thesis is dedicated to my parents; Noah Odek Opinde and Hilda Akinyi Opinde for their guidance and moral support since I started my education journey. iii ACKNOWLEDGEMENTS Thanks to Almighty God for this far He has brought us, his blessings and the gift of a healthy life. Despite the many challenges we have been through He has been good to us and we have been able to overcome them. I sincerely thank my supervisors; Dr. Anthony Kebira and Dr. Grace W. Gatheri for their relentless support, encouragement, relentless support and guidance during my research work. Special appreciation Professor L.E. Newton for the help and guidance offered in the process of identification of the plants. I would also like to dearly appreciate the support and input from my fellow colleagues; Philip Ogola, Japheth Wambani, Nathan Kiboi, James Kimani, Wycliffe Arika, Majorie Oruru, Christine Nakhumincha and Gilbert Koskey. May almighty God give you good health and continue blessing you in your endeavor’s throughout your lives. I acknowledge the support and assistance from Mr. Daniel Ng`ang`a Ms. Mary Njeri, Mr. Roy Mulanda, and the entire Microbiology Laboratory staff. May Almighty God shower you with his everlasting blessings, we made it at last. Finally, thanks to Almighty God again for in Him, we find the strength to explore new horizons; Amen. iv TABLE OF CONTENTS DECLARATION ..................................................................................................... i DEDICATION ........................................................................................................ ii ACKNOWLEDGEMENTS ................................................................................... iii TABLE OF CONTENTS ....................................................................................... iv LIST OF TABLES ............................................................................................... viii ABBREVIATIONS AND ACRONYMS .............................................................. ix ABSTRACT .............................................................................................................x CHAPTER ONE ......................................................................................................1 INTRODUCTION ...................................................................................................1 1.1 Background information ....................................................................................1 1.2 Problem statement ..............................................................................................4 1.3 Justification ........................................................................................................5 1.4 Research questions .............................................................................................6 1.5 Hypotheses .........................................................................................................7 1.6 Objectives of the study.......................................................................................7 1.6.1 General objective.........................................................................................7 1.6.2 Specific objectives.......................................................................................8 1.7 Significance of the study ....................................................................................8 CHAPTER TWO .....................................................................................................9 LITERATURE REVIEW ........................................................................................9 2.1 Medicinal plants .................................................................................................9 v 2.1.1 Tagetes minuta (Southern cone marigold) ..................................................9 2.1.2 Aloe secundiflora.......................................................................................11 2.1.3 Bulbine frutescens .....................................................................................12 2.1.4 Vernonia lasiopus ......................................................................................13 2.2 Test microorganisms ........................................................................................14 2.2.1 Escherichia coli .........................................................................................14 2.2.2 Salmonella typhi ........................................................................................15 2.2.3 Staphylococcus aureus ..............................................................................15 2.2.4 Candida albicans.......................................................................................16 2.2.5 Enterococcus faecalis ................................................................................17 2.2.6 Shigella flexineri .......................................................................................17 2.3 Phytochemicals in medicinal plants .................................................................18 2.3.1 Tannins ......................................................................................................19 2.3.2 Flavonoids .................................................................................................20 2.3.3 Alkaloids ...................................................................................................21 2.3.4 Saponins ....................................................................................................23 CHAPTER THREE ...............................................................................................25 MATERIALS AND METHODS ...........................................................................25 3.1 Plants sampling ................................................................................................25 3.2 Preparation of plant extract ..............................................................................25 3.3 Preparation of Media .......................................................................................26 3.3.1 Preparation of Potato dextrose agar ..........................................................26 vi 3.3.2 Preparation of Muller-Hinton agar ............................................................26 3.4 Preparation of susceptibility test discs .............................................................27 3.5 Test bacterial organisms ..................................................................................28 3.5.1 Isolation and Morphological identification ...............................................29 3.5.2 Biochemical identification ........................................................................31 3.5.3 Maintenance of the bacterial and fungal cultures......................................33 3.6 Antimicrobial susceptibility testing .................................................................33 3.7 Combined effect test ........................................................................................36 3.8 Qualitative phytochemical analysis .................................................................37 3.8.1Tannins .......................................................................................................37 3.8.2 Flavonoids .................................................................................................37 3.8.3 Alkaloids ...................................................................................................37 3.8.4 Saponins ....................................................................................................38 3.9 Statistical analysis ............................................................................................38 CHAPTER FOUR ..................................................................................................39 RESULTS ..............................................................................................................39 4.1 Efficacy test of the plant extracts on the bacterial pathogens and Candida albicans .............................................................................................39 4.2 Combined effect of the plant extracts on the bacterial pathogens and Candida albicans .............................................................................................47 4.3 Qualitative phytochemical analysis .................................................................52 CHAPTER FIVE ...................................................................................................53