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DETECTION OF BACTERIOPHAGE INFECTION USING ABSORBANCE, BIOLUMINESCENCE, AND FLUORESCENCE TESTS Thesis Submitted to The School of Engineering of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree Master of Science in Civil Engineering By Lindsey Marie Staley UNIVERSITY OF DAYTON Dayton, Ohio May, 2011 DETECTION OF BACTERIOPHAGE INFECTION USING ABSORBANCE, BIOLUMINESCENCE, AND FLUORESCENCE TESTS Name: Staley, Lindsey Marie APPROVED BY: ___________________________ __________________________ Denise Taylor, Ph. D., P.E. Kenya Crosson, Ph.D. Advisory Committee Chairman Committee Member Assistant Professor Assistant Professor Department of Civil and Department of Civil and Environmental Engineering and Environmental Engineering and Engineering Mechanics Engineering Mechanics ____________________________ Deogratias Eustace, Ph.D. Committee Member Assistant Professor Department of Civil and Environmental Engineering and Engineering Mechanics ___________________________ __________________________ John G. Weber, Ph.D. Tony E. Saliba, Ph.D. Associate Dean Wilke Distinguished Professor & School of Engineering Dean School of Engineering ii ABSTRACT DETECTION OF BACTERIOPHAGE INFECTION USING ABSORBANCE, BIOLUMINESCENCE, AND FLUORESCENCE TESTS Name: Staley, Lindsey Marie University of Dayton Advisor: Dr. Denise Taylor The activated sludge treatment process is a common method employed to treat wastewater. Normal operation of this process results in a floc-forming bacterial mixture, which settles rapidly. However, filamentous bacteria can cause sludge bulking, which interferes with the compaction and settling of flocs. A common method to control sludge bulking is adding a chemical such as chlorine to the activated sludge basin, which kills not only the problematic bacteria, but also the essential floc-forming bacteria. Bacteriophages (phages) are viruses that only infect bacteria. It is hypothesized that phages of filamentous bacteria can be added to the activated sludge basin to control sludge bulking, rather than a chemical. Due to the unique morphology of filamentous bacteria, traditional methods such as the plate method do not work well to detect phage infection. The purpose of this thesis was to detect infection of bacteria by phages using absorbance, bioluminescence, and fluorescence broth tests. E. coli and T2 iii phage was first used to establish a model of the bacteria-phage relationship using these tests. All three broth methods show evidence of phage infection in T2 phage and E. coli mixtures. Following this, phages were isolated from activated sludge systems and were applied to E. coli and S. natans, an example of filamentous bacteria found in activated sludge bulking problems. Their growth patterns were observed using the above mentioned tests. E. coli showed obvious infection patterns, but S. natans test sets were highly variable and phage infection patterns could not be distinguished. For the absorbance test and fluorescence test, ratios of bacteria to phage that clearly showed phage infection were 1000:1 and 100:1. Low concentrations of bacteria (i.e. 105 cfu/mL) are recommended for use when preparing samples because even if the titer of phage from an environmental sample is higher than expected, phage infection patterns will still be detected. The bioluminescence test showed infection patterns for all ratios of bacteria to phage. The concentration of bacteria used to prepare these ratios did not affect these patterns. The bioluminescence test is recommended to detect infection of bacteria by phages from activated sludge samples for these reasons. iv ACKNOWLEDGEMENTS I would like to express my deep appreciation to Dr. Denise Taylor for her guidance and patience throughout this research project. I would also like to thank the Ohio Water Development Authority for funding and the opportunity to work on this project. v TABLE OF CONTENTS ABSTRACT ..................................................................................................iii ACKNOWLEDGEMENTS .............................................................................v LIST OF FIGURES .......................................................................................x LIST OF TABLES .........................................................................................xv CHAPTER I. INTRODUCTION ..........................................................................1 1.1 Problem Statement ................................................................1 1.2 Study Objectives ....................................................................2 1.3 Thesis Organization ...............................................................3 II. LITERATURE REVIEW ................................................................4 2.1 Sludge Bulking ........................................................................4 2.2 Control of Sludge Bulking .......................................................5 2.3 Filamentous Bacteria ..............................................................6 2.4 Bacteriophage History ............................................................7 2.5 Bacteriophage Biology ............................................................9 2.6 Isolation of Bacteriophages from Activated Sludge ................11 2.7 Applications of Bacteriophage ................................................11 2.8 Detection of Bacteriophages using Absorbance, Bioluminescence, and Fluorescence Tests ............................13 2.9 Literature Review Summary ...................................................15 III. MATERIALS AND METHODS ......................................................17 3.1 Materials .................................................................................17 vi 3.1.1 Bacteria and Bacteriophages .......................................20 3.1.2 Media............................................................................21 3.2 Methods ..................................................................................21 3.2.1 Quantification of Cell Density .......................................21 3.2.2 Propagation and Harvesting of T2 Phage ....................22 3.2.3 Titer of T2 Phage ..........................................................23 3.2.4 Collection and Filtration of Wastewater Samples for Isolation of Phages .......................................................24 3.2.5 Growth of Sphaerotilus natans .....................................25 3.2.6 Preparation of 12 Well Multiwell™ Plate for Use in Absorbance, Bioluminescence, and Fluorescence Tests for E. coli and T2 Phage .....................................26 3.2.7 Preparation of 12 Well Multiwell™ Plate Used in Absorbance, Bioluminescence, and Fluorescence Test for E. coli and E. coli Phage Enrichment ..............27 3.2.8 Preparation of 12 Well Multiwell™ Plate Used in Absorbance, Bioluminescence, and Fluorescence Test for S. natans and S. natans Phage Enrichment ....28 3.2.9 Absorbance Test of Phage Infection ............................29 3.2.10 BacTiter-Glo™ Microbial Cell Viability Assay............................................................................31 3.2.10.1 Preparation of BacTiter-Glo™ Reagent ........31 3.2.10.2 Bioluminescence (ATP) Test ........................32 3.2.11 L7012 LIVE/DEAD® BacLight™ Bacterial Viability Kit .........................................................33 3.2.11.1 Preparation of L7012 LIVE/DEAD® BacLight™ Stain ...........................................34 3.2.11.2 Fluorescence (Membrane)Test ....................34 3.2.12 Enrichment of Phage Filtrate with S. natans.................35 3.2.13 Enrichment of Phage Filtrate with E. coli ......................36 3.2.14 Spot Test of Phage Enrichment on S. natans Lawn .....37 3.2.15 Collection of Phage Plaques and Phages from Spot Test of Phage Enrichment on S. natans .......................38 3.2.16 Titration of Filtered and Non-filtered T2 Phage .............38 3.2.17 Titration of T2 Phage Introduced to Hard Agar .............40 3.2.18 Growth Curve of S. natans using Absorbance ..............41 3.2.19 Absorbance Test on E. coli and BacTiter-Glo ™ Reagent Kept at Room Temperature ...........................42 3.2.20 E. coli and BacTiter-Glo Reagent™ Dilutions Absorbance Tests ........................................................43 vii 3.2.21 Absorbance Tests of Mixed Microbial Community with Different Diluents ..........................................................44 IV. RESULTS AND DISCUSSION .....................................................45 4.1 Absorbance Test Results for E. coli and T2 Phage ...............45 4.2 Bioluminescence (ATP) Test Results for E. coli and T2 Phage ...............................................................................54 4.3 Fluorescence (Live/Dead) Test Results for E. coli and T2 Phage ...............................................................................61 4.4 Comparison of Absorbance, Bioluminescence, and Fluorescence Tests for E. coli and T2 Phage .......................69 4.5 Summary of Collected Samples of Activated Sludge .............71 4.6 Absorbance Test Results for E. coli and “Troy 2 E. coli Enrichment 1” ........................................................................73 4.7 Bioluminescence Test Results for E. coli and “Troy 2 E. coli Enrichment 1” ........................................................................77 4.8 Fluorescence Test Results for E. coli and “Troy 2 E. coli Enrichment 1” ........................................................................80
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