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Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys Olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties Erin L

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Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys Olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties Erin L Indiana University – Purdue University Fort Wayne Opus: Research & Creativity at IPFW Master's Theses Master's Theses and Graduate Research 8-1-2012 Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties Erin L. Keene Indiana University - Purdue University Fort Wayne Follow this and additional works at: http://opus.ipfw.edu/masters_theses Part of the Biology Commons, Environmental Microbiology and Microbial Ecology Commons, and the Marine Biology Commons Recommended Citation Erin L. Keene (2012). Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties. http://opus.ipfw.edu/masters_theses/19 This Master's Research is brought to you for free and open access by the Master's Theses and Graduate Research at Opus: Research & Creativity at IPFW. It has been accepted for inclusion in Master's Theses by an authorized administrator of Opus: Research & Creativity at IPFW. For more information, please contact [email protected]. Graduate School ETD Form 9 (Revised 12/07) PURDUE UNIVERSITY GRADUATE SCHOOL Thesis/Dissertation Acceptance This is to certify that the thesis/dissertation prepared By Erin L. Keene Entitled Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties Master of Science For the degree of Is approved by the final examining committee: Frank V. Paladino Chair Tanya T. Soule Elliott J. Blumenthal To the best of my knowledge and as understood by the student in the Research Integrity and Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material. Approved by Major Professor(s): ____________________________________Frank V. Paladino ____________________________________ Approved by: Frank V. Paladino 07/20/2012 Head of the Graduate Program Date Graduate School Form 20 (Revised 9/10) PURDUE UNIVERSITY GRADUATE SCHOOL Research Integrity and Copyright Disclaimer Title of Thesis/Dissertation: Microorganisms from Sand, Cloacal Fluid, and Eggs of Lepidochelys olivacea and Standard Testing of Cloacal Fluid Antimicrobial Properties For the degree of MasterChoose ofyour Science degree I certify that in the preparation of this thesis, I have observed the provisions of Purdue University Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.* Further, I certify that this work is free of plagiarism and all materials appearing in this thesis/dissertation have been properly quoted and attributed. I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the United States’ copyright law and that I have received written permission from the copyright owners for my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless Purdue University from any and all claims that may be asserted or that may arise from any copyright violation. Erin L. Keene ______________________________________ Printed Name and Signature of Candidate 07/18/2012 ______________________________________ Date (month/day/year) *Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html MICROORGANISMS FROM SAND, CLOACAL FLUID, AND EGGS OF Lepidochelys olivacea AND STANDARD TESTING OF CLOACAL FLUID ANTIMICROBIAL PROPERTIES A Thesis Submitted to the Faculty of Purdue University by Erin L. Keene In Partial Fulfillment of the Requirements for the Degree of Master of Science August 2012 Purdue University Fort Wayne, Indiana ii ACKNOWLEDGMENTS I want thank all the people who made this project possible. First, I would like to start by thanking my amazing committee who answered all the questions I had throughout the project and even took the time to visit my laboratory in Costa Rica. This includes my advisor, Dr. Frank Paladino, who not only gave me the freedom to choose any research topic I desired for my thesis, but also the freedom to design my own study and the necessary tools, guidance, and financial necessary to complete this project. Also, I want to thank Dr. Tanya Soule who came in half-way through the project but quickly became an indispensable member of the committee. Her experience and insight into the field of microbiology allowed me to be far more successful during the second half of my project. My third committee member, Dr. Elliot Blumenthal, was also an amazing mentor and answered any questions I had. Besides my committee, I would like to thank all the people who helped me prepare my lab and complete my work, as well as MINAE, who approved my permits for the research. Bruce, Arlis, and Dar made sure I had all of the supplies and paperwork I needed in Costa Rica, not an easy task considering they were in Indiana. The School of Veterinary Medicine at Universidad Nacional de Costa Rica was so kind in sending me supplies, especially a much-needed alcohol lamp, as well a copy of a thesis from another student who had isolated bacteria and fungi from nesting beaches, but had who not iii published their work. I would also like to thank the Department of Biology at The University of Puget Sound, in Tacoma, WA for sending supplies with my parents when they came to visit. As for Dr. Carlos Núñez Navas at Laboritorio Clínico y Banco de Sangre San Jose, there is not enough thanks in the world for how much you helped me with my identifications and locating the bacteria for my antimicrobial assays. I would also like to thank everyone associated with The Leatherback Trust, especially Dr. “Bibi” Santidrían Tomillo who procured my permits, dealt with my less than perfect Spanish on important government documents, and allowed me to stay at the marine station for free for a second year, even though I was not a member of the field team. I apologize my additional month there turned into six. Thank you, Dr. Jim Spotila, for letting me hang around for an extra year as well, your project advice, and the M&Ms. I would also like to thank the 2010-2011, and 2011-2012 field teams for always eagerly helping collect samples (I would name you individually, but there are too many). Also, thanks to the scores of Earthwatch volunteers who, through The Leatherback Trust patrolled with us, allowing me an occasional night off to sleep and do lab work. Finally, thanks to all my family and friends who supported me throughout my three years of graduate school. I apologize for all of the holidays, birthdays, weddings, and graduations I missed, but I was there in spirit. Thank you Noreen for being neighbor of the year and helping me translate my proposal-two years in a row! Thank you to every graduate student who allowed me to crash on your couch because I did not want to move to Indiana. Sorry, mom and dad, that you could not make it to the beach more often (and that mom did not get to see a turtle), but thanks for trying multiple times! Also, the team and I really appreciated the chocolate and gifts you sent. Love you all! iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES ......................................................................................................... viii ABSTRACT .........................................................................................................................x CHAPTER 1: INTRODUCTION TO STUDY ...................................................................1 Study Species ...........................................................................................................2 Study Sites ...............................................................................................................4 Monitoring Sea Turtles ............................................................................................9 Olive Ridley Egg Development .............................................................................11 Bacteria and Fungi in Eggs ....................................................................................14 Sterilization ............................................................................................................17 Bibliography ..........................................................................................................19 CHAPTER 2: ISOLATION OF BACTERIA FROM SEA TURTLE NEST CHAMBER SAND, OVIDUCTAL FLUIDS, AND UNHATCHED EGGS .....................................................................................................................22 Introduction ............................................................................................................22 Methods..................................................................................................................28 Results ....................................................................................................................33 Discussion ..............................................................................................................39 Bibliography ..........................................................................................................50 CHAPTER 3: ISOLATION OF FUNGI FROM SEA TURTLE NEST CHAMBER SAND, OVIDUCTAL FLUIDS, AND UNHATCHED EGGS .....................................................................................................................55 Introduction
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