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

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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.

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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.

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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

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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

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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 4.9 Summary of Broth Tests Involving E. coli and Phage Source ...... 84 4.10 Growth of S. natans ...... 85 4.11 Tracking the Growth Curve of S. natans Using Absorbance...... 86 4.12 Absorbance Test Results for S. natans and “Troy 2 S. natans Enrichment 1 P1” ...... 88 4.13 Bioluminescence Test Results for S. natans and “Troy 2 S. natans Enrichment 1 P1” ...... 91 4.14 Fluorescence Test Results for S. natans and “Troy 2 S. natans Enrichment 1 P1” ...... 93 4.15 Summary of Broth Tests Involving S. natans and Phage Source ...... 96 4.16 E. coli and BacTiter-Glo™ Reagent Absorbance Test ...... 97 4.17 E. coli and BacTiter-Glo™ Reagent Dilutions Absorbance Tests ...... 99 4.18 Retrieving Phage from a Sample Containing BacTiter-Glo or Live/Dead Stain ...... 101 4.19 Absorbance Tests of Mixed Microbial Community with Different Diluents ...... 102 4.20 Effects of Filtering on T2 Phage Titer ...... 106

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4.21 Retrieving T2 Phage from Hard Agar ...... 108

V. CONCLUSIONS AND RECOMMENDATIONS ...... 110

5.1 Isolation of Phages from Activated Sludge ...... 110 5.2 Detection of Phage Infection Using the Absorbance Test ...... 111 5.3 Detection of Phage Infection Using the Bioluminescence Test ...... 112 5.4 Detection of Phage Infection Using the Fluorescence Test ...... 113 5.5 Next Research Steps ...... 114

REFERENCES ...... 116

APPENDICES

A. SYNERGY 4™ AND GEN5™ PROTOCOLS ...... 120

B. ABSORBANCE DATA FOR E.COLI AND T2 PHAGE ...... 122

C. DATA FOR BIOLUMINESCENCE TESTS FOR E. COLI AND T2 PHAGE ...... 128

D. DATA FOR FLUORESCENCE TESTS FOR E. COLI AND T2 PHAGE ...... 133

E. DATA FOR E. COLI AND “TROY2 E. COLI ENRICHMENT 1” ...... 138

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LIST OF FIGURES

3.1 McFarland Equivalence Turbidity Standards Used to Estimate Cell Density ...... 22

3.2: Layout of the 12 Well Plate for E. coli and T2 Phage ...... 26

3.3: Layout of the 12 Well Multiwell™ Plate for E. coli and E. coli Phage Enrichment ...... 28

3.4: Layout of the 12 Well Multiwell™ Plate for S. natans and S. natans Phage Enrichment ...... 29

3.5: Layout of Spot Test ...... 37

3.6: Plate Layout for E. coli and BacTiter-Glo Assay Absorbance Tests ...... 43

4.1: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at Room Temperature ...... 50

4.2: Absorbance Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 51

4.3: Absorbance Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 51

4.4: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 51

4.5: Absorbance Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 52

4.6: Absorbance Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 52

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4.7: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 52

4.8: Comparisons of Ratios of E. coli to Phage ...... 53

4.9: Comparison of the 1000:1 E. coli: Phage Ratio ...... 53

4.10: Comparison of the 10000:1 E. coli: Phage Ratio ...... 53

4.11: ATP Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 58

4.12: ATP Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 58

4.13: ATP Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 58

4.14: (a) ATP Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 (b) Zoomed to Show Initial ATP Increase of Phage Samples ...... 59

4.15: (a) ATP Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 (b) Zoomed to Show Initial ATP Increase of Phage Samples ...... 59

4.16: ATP Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 59

4.17: Normalized Data of Figure 4.11 ...... 60

4.18: Normalized Data of Figure 4.12 ...... 60

4.19: Normalized Data of Figure 4.13 ...... 60

4.20: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 30°C, Trial 1 ...... 65

4.21: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 30°C, Trial 1 ...... 65

4.22: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 30°C, Trial 2 ...... 66

4.23: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 30°C, Trial 2 ...... 66

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4.24: Live/Dead Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 67

4.25: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 67

4.26: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1 ...... 67

4.27: Live/Dead Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 68

4.28: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 68

4.29: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 ...... 68

4.30: Comparisons of Different E. coli to T2 Phage Ratios for Live/Dead Test ...... 69

4.31: Absorbance Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1 ...... 75

4.32: Absorbance Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1 ...... 75

4.33: Absorbance Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1 ...... 75

4.34: Absorbance Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2 ...... 76

4.35: Absorbance Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2 ...... 76

4.36: Absorbance Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2 ...... 76

4.37: (a) ATP Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage and (b) Zoomed View to Show Early Increase in ATP ...... 79

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4.38: (a) ATP Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage and (b) Zoomed View to Show Early Increase in ATP ...... 79

4.39: ATP Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source ...... 79

4.40: Live/Dead Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source ...... 83

4.41: Live/Dead Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source ...... 83

4.42: Live/Dead Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source ...... 83

4.43: S. natans Growth Curve Using Sweep Read Mode ...... 87

4.44: S. natans Growth Curve Using Normal Read Mode ...... 87

4.45: Absorbance Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 89

4.46: Absorbance Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 89

4.47: Absorbance Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 89

4.48: Absorbance Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 90

4.49: Absorbance Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 90

4.50: Absorbance Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 90

4.51: ATP Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source ...... 92

4.52: ATP Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source ...... 92

4.53: ATP Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source ...... 92

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4.54: Live/Dead Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 94

4.55: Live/Dead Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 94

4.56: Live/Dead Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1 ...... 94

4.57: Live/Dead Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 95

4.58: Live/Dead Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 95

4.59: Live/Dead Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2 ...... 95

4.60: Control Absorbance Curve for E. coli + Sterile Water ...... 98

4.61: Absorbance Curve for E. coli + BacTiter-Glo Reagent Kept at Room Temperature for 96 Hours ...... 98

4.62: E. coli and BacTiter-Glo Dilutions Growth Curves (Sample – Blank) ...100

4.63: Absorbance of Blanks over Time ...... 100

4.64a: Growth Curves for Different Concentrations of Supernatant to Sterile Water (Sample – Blank) ...... 103

4.64b: Expanded Window of Growth of Supernatant and Sterile Water ...... 103

4.65a: Growth Curves for Different Concentrations of Supernatant to Nutrient Broth + 0.5% NaCl (Sample – Blank) ...... 104

4.65b: Expanded Window of Growth of Supernatant and Nutrient Broth + 0.5% NaCl ...... 104

4.66a: Growth Curves for Different Concentrations of Supernatant to CYGA (Sample – Blank) ...... 105

4.66b: Expanded Window of Growth of Supernatant and CYGA ...... 105

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LIST OF TABLES

3.1: Materials ...... 17

3.2: Equipment ...... 19

3.3: Chemical ...... 20

4.1: Activated Sludge Samples and Enrichments ...... 72

4.2: Data collected from Titer of T2 Phage ...... 106

4.3: Hypothesis Test Results ...... 107

4.4: One-Sample T-test Statistics ...... 107

4.5: One-Sample T-Test Results ...... 108

4.6: Titer of T2 Phage in Hard Agar ...... 109

4.7: Titer of T2 Phage Stock without Hard Agar ...... 109

B1: Absorbance Data for Jan 19 2010 ...... 122

B2: Absorbance Data for Jan 21 2010 ...... 123

B3: Absorbance Data for Jan 28 2010 ...... 124

B4: Absorbance Data for Sept 17 2010 ...... 125

B5: Absorbance Data for Sept 22 2010 ...... 126

B6: Absorbance for Data Oct 4 2010 ...... 127

C1: Bioluminescence Data for Jan 19 2010 ...... 128

C2: Bioluminescence Data for Jan 21 2010 ...... 129

C3: Bioluminescence Data for Jan 28 2010 ...... 130

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C4: Bioluminescence Data for Sept 17 2010 ...... 131

C5: Bioluminescence Data for Oct 4 2010 ...... 132

D1: Fluorescence Data for Feb 4 2010 ...... 133

D2: Fluorescence Data for Feb 22 2010 ...... 134

D3: Fluorescence Data for Mar 2 2010 ...... 135

D4: Fluorescence Data for Sept 22 2010 ...... 136

D5: Fluorescence Data for Oct 4 2010 ...... 137

E1: Absorbance Data for Oct 24 2010 ...... 139

E2: Absorbance Data for Nov 12 2010 ...... 140

E3: Bioluminescence Data for Oct 24 2010 ...... 141

E4: Fluorescence Data for Nov 12 2010 ...... 142

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CHAPTER I

INTRODUCTION

1.1 Problem Statement

Sludge bulking caused by excessive growth of filamentous bacteria is a problem encountered in activated sludge processes in wastewater treatment plants. Sludge bulking causes poor settleability of biological flocs, which can lead to these flocs being discharged into the environment. A common way to control sludge bulking is to add a chemical to kill the filamentous bacteria, but this method kills all types of bacteria, including those that are essential to degradation of organic material. For this reason, new methods of controlling filamentous bacteria are highly desirable. Biocontrol of filamentous bacteria through bacteriophage (phage) is one of these new methods being investigated.

Since filamentous bacteria grow in sheaths and are not dispersed homogeneously throughout a culture, traditional methods, such as the plate method, used to detect phage infection of bacteria are not efficient because the filamentous bacteria are not evenly distributed throughout the agar. Phages that are located in an area without bacteria will not be detected via plaque formation.

A more efficient method to detect phage of filamentous bacteria is desirable.

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1.2 Study Objectives

The objective of the multiyear project of which this thesis is a part is to isolate a phage specific to Sphaerotilus natans, which is the model filamentous bacteria chosen for the project, using the absorbance, bioluminescence, and fluorescence tests, and to use this phage to reduce sludge bulking induced by S. natans. Since the plate method is not a practical detection method for phages that infect filamentous bacteria, the absorbance, bioluminescence, and fluorescence broth tests are to be explored to detect phage infection. Due to the morphology of S. natans, a reliable model of phage infection using these tests is first needed. Results from tests performed with S. natans and a potential phage source can be compared to the model to determine if phages of S. natans are present in the phage source. E. coli and T2 phage were chosen as a model because E. coli is robust, easy to grow, and yields repeatable results.

Previous work on the multiyear project showed that phage infection of E. coli by T2 phage could be detected through the absorbance test. It also suggested which concentrations and ratios of E. coli and T2 phage best demonstrated signs of phage infection through the absorbance test (Li 2009).

The portion of this project addressed in this thesis is the detection of patterns that indicate phage infection, using E. coli and T2 phage as a model.

The detection of these patterns were monitored by absorbance, ATP (adenosine triphosphate), and membrane integrity tests. This thesis also discusses the determination of which ratios of bacteria to phage best show phage infection in these tests. The applicability of these patterns and ratios to detect the presence

2 of phages in samples collected from activated sludge processes that are infective against E. coli and S. natans is also addressed.

1.3 Thesis Organization

In Chapter 2, literature on sludge bulking causes and control, bacteriophages, filamentous bacteria, and the broth tests is reviewed. The methods used throughout the research performed for this thesis can be found in

Chapter 3.

Chapter 4 contains the results and discussion of the research performed.

First, the results from tests with E. coli and T2 phage are presented and discussed, followed by a comparison between the three tests for this model.

Next, the E. coli and phage source test results follow, and are discussed and compared to each other as well as to the E. coli and T2 phage model. After these sections, work with S. natans and possible S. natans phages isolated from activated sludge can be found. Finally, tests regarding the effects of isolation procedures on the titer of phages is presented and discussed.

Conclusions drawn from this thesis as well as recommendations for future work are found in Chapter 5. The recommendations include how phages should be isolated from activated sludge, as well as what patterns should be detected in the absorbance, bioluminescence, and fluorescence tests that would imply phages of the host bacteria are present. Finally, protocols used for the Synergy

4™ machine and Gen5™ software, as well as data measurements are offered in the appendices.

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CHAPTER II

LITERATURE REVIEW

2.1 Sludge Bulking

The activated sludge treatment process is a commonly used method in municipal and industrial wastewater treatment plants and employs biological decomposition to treat and reduce the biochemical oxygen demand (BOD) of wastewater. Normal operation of the activated sludge process results in a floc- forming bacterial mixture, which dominates the population, which is responsible for oxidation of organic materials, and settles rapidly. However, filamentous bacteria can cause sludge bulking and foaming, which interferes with the compaction and settling of flocs (Wagner et al. 2002).

In order for an activated sludge process to operate efficiently, the sludge particles (flocs) must be able to settle out. When there is excessive growth of filamentous bacteria such as Sphaerotilus natans, sludge bulking occurs. During sludge bulking, filamentous bacteria grow excessively in the filamentous phase inside of a floc, which provides structure to which zoogleal, or floc-forming, microorganisms can attach via capsular or polysaccharides which they produce

(Horan et al. 1988). This web extending out of the floc can catch other bacteria, colloids and compact flocs that are moving toward the bottom of the sedimentation basin. This process results in a fluffy, non-compact floc which

4 does not settle well and can ultimately be discharged out of the top of the sedimentation basin and into the receiving body of water (Horan et al. 1988).

Occurrence of sludge bulking is typically attributed to the following conditions in an aeration basin: low dissolved oxygen, insufficient nutrients, surplus sulfide wastes, small food-to-microorganism ratio, and low pH (Jenkins and Richard 1985). A commonly used indicator of sludge bulking is the sludge volume index, or SVI, which is a measure of the sludge settleability. The threshold for the occurrence of sludge bulking is generally agreed to be 150 mL/g

(Schuler and Jassy 2007). Under these conditions, filamentous bacteria begin to grow in the filamentous phase, in which the bacteria are strung together in long chains inside sheaths. These sheaths extend out of the floc and catch other flocs that are settling toward the bottom, as described above. When the sum of the lengths of each of these filaments in one milliliter of activated sludge is in excess of 107 µm, it is a good indicator of sludge bulking (Palm et al. 1980).

2.2 Control of Sludge Bulking

When sludge bulking occurs, one way to kill filamentous bacteria is to add a chemical such as chlorine, but the chemical kills the crucial floc-forming bacteria as well (Kitatsuji et al. 1996), which could be detrimental to the treatment process. Control of sludge bulking via chlorine can also create disinfection by- products, which cause serious health problems. The presence of high suspended solids and nitrite will reduce the effectiveness of chlorine at

5 controlling sludge bulking (Neethling et al. 1985). For these reasons, a method that kills only the targeted bacteria without harmful side effects is needed.

Operational parameters can also be altered to attempt to control sludge bulking. If sludge bulking is caused by a low food to microorganism ratio, raising the dissolved oxygen can reduce the filament length and lower the sludge volume index, indicative of improved settleability. The feed patterns can also be altered to control the food to microorganism (F:M) ratio (Palm et al. 1980). The relationship between dissolved oxygen and the food to microorganism ratio established by Palm et. al (1980) is linear, so the higher the F:M ratio, the more dissolved oxygen is required. This can greatly increase energy costs associated with the generation of oxygen. In addition, the change from bulking to non- bulking conditions can longer than control with chemicals.

2.3 Filamentous Bacteria

There are more than 30 different types of filamentous bacteria present in the activated sludge process (Nielsen et al. 2009). Under normal conditions, filamentous bacteria provide a structure of filaments to which flocculent bacteria can attach, and a dense floc is formed which can quickly settle out. Under bulking conditions, the floc becomes less dense, and therefore the settleability is reduced.

In conventional wastewater treatment plants that treat municipal wastewater, most of the filamentous bacteria consume complex compounds such as proteins and lipids, which floc-forming bacteria are unable to degrade, and are

6 highly resistant to starvation (Nielsen et al. 2009). Because of this, these type of filamentous bacteria are difficult to control by changing operational parameters such as F:M ratio and dissolved oxygen levels (Nielsen et al. 2009).

When filamentous bacteria grow in the filamentous phase, the bacteria are encapsulated in a sheath. This causes the bacteria to grow in clumps and to be distributed in a non-homogenous fashion throughout the media. Because of this, when grown in a culture, filamentous bacteria will not be homogenously distributed throughout the growth media, unlike non-filamentous bacteria, such as E. coli. Due to this morphology, filamentous bacteria must be broken up via homogenization.

2.4 Bacteriophage History

Bacteriophages (phages) are viruses that only infect bacteria.

Bacteriophages, which exist in the estimated range of 1030 to 1032 different types, are the most profuse organisms known to man. Phages are an integral part of the stability of environmental microbial communities and are important to keeping bacterial populations in check (Kutter and Sulakvelidze 2005). The discovery of phage is credited to Frederick Twort in 1915 and Felix d’Herelle in 1917. Twort observed mucoid, “glassy” colonies in colonies of bacteria, which could be introduced into other bacteria colonies and cause the same “glassy transformation,” and that this process could be easily replicated (Summers

2005). After examining the bacteria from the “glassy” colonies, Twort noticed that the bacteria had disintegrated into small pieces. He then hypothesized that

7 infectious viruses that were smaller than bacteria were causing this transformation to occur (Summers 2005).

D’Herelle observed that what he termed “bacteriophage” caused lysis, or rupture, of bacteria in liquid as well as in clear spots on agar, which he called

“plaques.” He assumed that bacteriophages were parasites of bacteria. Through his work, he observed that phages need host cells (bacteria) in order to reproduce, and that reproduction could occur for an indefinite period, but lysis of the host cells was a necessary part of the reproduction process. D’Herelle showed that counting the number of plaques formed on a bacteria culture in agar provided a way to estimate the number of bacteriophage present in a given aliquot. He also noted that phage reproduced in cycles in which hosts were infected by phages, and then phages were produced inside the host then released. The phages then infected new cells and the cycle was repeated

(Summers 2005).

In the 1930s, phages were purified and shown to be made of proteins and phosphorus. In 1936, Max Schlesinger showed that phages contain DNA through the Feulgen reaction, which is a staining technique used to identify DNA

(Summers 2005).

The invention of the electron microscope in the 1940s allowed scientists to observe phages and the relationship between phages and bacteria. It also proved that different species of phages have different morphologies, as well as confirming that phages contain DNA. In 1952, Hershey and Chase showed that when a phage attaches to a host cell, its DNA is injected into the cell and the

8 phage’s protein coat does not enter the cell and does not affect phage reproduction (Summers 2005).

2.5 Bacteriophage Biology

Phages need host bacteria to reproduce because they only carry DNA or

RNA from which more phages can be produced once the genes are injected into an applicable host. Phages cannot produce energy and do not contain ribosomes to produce proteins; however, phages do remain viable outside of a host for up to decades, as long as they are not damaged (Guttman et al. 2005).

Many factors can affect the viability of phages. All phages are sensitive to

UV light and are inactivated by it, including sunlight and fluorescent light. Other things that damage phages include pH (phages are usually stable between pH 5 and 8, even down to 3 to 4), protein-denaturing agents such as urea, detergents, heat, and alcohols. Chloroform, which can be used to lyse bacteria cells containing phages, does not generally interact with non-enveloped phages, which do not have the extra protective layer that enveloped phages do (Guttman et al. 2005).

Many phages are specific to the bacteria it can infect, which may include subspecies of one species of bacteria (Synnott et al. 2009). This is because most phages are only capable of interacting with receptors of host bacteria that have a specific structure in order to inject DNA. Some species of phages, however, are able to infect multiple species of bacteria; these are known as broad-host-range phages (Jensen et al. 1998; Guttman et al. 2005).

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Phages are classified based on whether they are virulent or temperate.

Virulent phages can only reproduce via a lytic cycle. In the lytic cycle, the phage attaches to the host cell and injects its DNA, from which more phages are produced by mechanisms inside the host cell. After reproduction of phages, the host cell lyses and releases the phages into the environment. Virulent phages typically program proteins in their host cells which are deadly. These proteins change the ability of the host cell to reproduce or transcribe DNA, alter the host’s

DNA, degrade or redirect enzymes, and change the host’s membrane (Guttman et al. 2005).

Temperate phages, on the other hand, do not automatically induce a lytic cycle inside the host cell. The phage may induce a lysogenic cycle in which the

DNA enters a dormant state, called a prophage. When the host cell reproduces, the prophage is also included in the new cell. The prophage has the ability to come out of the inert state and enter a lytic cycle if stress or damage is induced on the host cell (Guttman et al. 2005).

When temperate prophages are in the lysogenic state, a symbiotic relationship between the bacterium and phage occurs. The prophage encodes a repressor protein into the host cell, which blocks the transcription of phage genes. This repressor also prevents genes of other phages that may infect the host cell from being transcribed, which protects the cell from death by lysis. The prophage can also increase the bacterium’s resistance to antibiotics (Guttman et al. 2005).

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Because temperate phages do not immediately enter the lytic phase once the DNA enters a bacterium, these types of phages are not desirable for biocontrol of filamentous bacteria in activated sludge. Virulent phages are necessary to cause lysis of the bacteria within a few hours of introduction and thus reduce sludge bulking quickly.

2.6 Isolation of Bacteriophages from Activated Sludge

There are instances where phages specific to a bacteria of interest were isolated from activated sludge processes and successfully used for biocontrol of a specific host (Kotay et al. 2011; Synnott et al. 2009). This implies that phages of S. natans can be isolated from activated sludge as well. In the mixed liquor suspended solids of an activated sludge process, total phages are present in a range of 104 to 109 pfu/mL (plaque-forming-units per milliliter) (Withey et al.

2005). Phages of common fast-growing floc-forming bacteria, such as coliphages (phages of E. coli) are present in concentrations of 103 to 104 pfu/mL

(Tanji et al. 2003). With slower growing bacteria like filamentous bacteria, however, the concentration of phages that infect these bacteria may be lower and thus harder to isolate

2.7 Applications of Bacteriophage

The use of phages for controlling bacterial growth is gaining attention in the food industry due to studies showing the effective use of phages to biocontrol bacteria that cause food-borne illnesses. In 2007, the Food and Drug

11

Administration approved the addition of phages to food to control the potentially deadly bacteria Listeria monocytogenes in ready-to-eat foods. This marked the first time phages had been approved as a food additive (Bren 2007). Such approval has perhaps sparked even more interest in phages as food additives to control other harmful bacteria. In one study, phages specific to E. coli were shown to reduce E. coli contamination of broccoli, tomatoes, spinach, and ground beef an average of 98.5%, 97%, 99.5%, and 94.5% respectively (Abuladze et al.

2008).

The use of phages to control the growth of bacteria is also increasing in interest in the medical field due to the increased cases of antibiotic-resistant bacteria. One such bacterium is Staphylococcus aureus, which causes the disease bovine mastitis in cows in addition to many other diseases in humans and animals. Synnott et al. (2009) isolated phages of S. aureus from activated sludge and tested their ability to lyse S. aureus by the plate method, and ultimately selected two of these phages and tracked the growth of a mixture of S. aureus and these phages over time using absorbance. These tests revealed that the phages caused a decrease in absorbance, and the authors attributed this decrease to lysis of S. aureus by the phages (Synnott et al. 2009). By using phages to kill bacteria instead of antibiotics, the demand for antibiotics may decrease, which may slow the development of antibiotic-resistant strains of bacteria.

Recently, the first demonstration of biocontrol of filamentous sludge bulking using phages was performed by Kotay et al. (2011). In order to identify

12 phages from an activated sludge sample, Kotay et al. (2011) employed the top- agar plating technique using the filamentous bacteria Haliscomenobacter hydrossis and a phage extract concentrated from an activated sludge sample.

One phage was isolated from this technique, and was called “HHY-phage.” This

HHY-phage was purified via the repeat plaque assay, and the titer was calculated to be about 5 x 105 pfu/mL. It was also determined that the HHY- phage is a lytic phage that induces lysis of H. hyrossis and causes infected bacteria to lyse 30 minutes after adsorption, with a burst size of about 105 pfu/infected cell. HHY-phage was found not to be infective to several bacteria that play important roles in BOD and nutrient removal.

After these important properties of this phage were established, it was applied to a mixture of activated sludge and H. hydrossis at a ratio of 1000:1 host to phage. The SVI of the sample containing phage was reduced from 156, which is indicative of sludge bulking, to 105. This SVI is below the sludge bulking threshold, and the reduction was attributed to the phage lysing the host filamentous bacteria.

2.8 Detection of Bacteriophages using Absorbance, Bioluminescence, and

Fluorescence Tests

Since phages are not visible to the eye, even with a microscope, due to their small size, evidence of infection of bacteria by phages is monitored through tests. A standard method for determining infection of bacteria with phages is the plate method. In this method, a mixture of bacteria, phages, growth media and

13 agar is poured onto a hard agar plate. When the phages infect the bacteria and cause the cells to lyse, plaques, or clear spots, are formed in the agar that is cloudy due to growth of bacteria. This method does not work well for bacteria that are not homogeneously dispersed throughout the mixture, such as filamentous bacteria, so a different method for identifying infection must be used.

Absorbance, bioluminescence, and fluorescence tests will be used to provide evidence of infection. E. coli and T2 phage was chosen as a model because E. coli is robust, easy to grow, and reliable. S. natans, the chosen filamentous model, requires more particular growth conditions and much more time to grow, which is why a reliable model was needed first.

The absorbance test detects the amount of light that is not blocked by particles in the material through which it is traveling. As bacteria multiply, the amount of light blocked will increase. As cells lyse, or rupture, due to infection by phages, the amount of light that can pass through the sample will increase, resulting in a decrease in absorbance. This pattern was shown in a study by

Synnott et al. (2009), which was discussed above.

The bioluminescence test detects the amount of ATP, or cell energy, present in a sample using a reagent that interacts with ATP and emits light. The light emitted during the test is proportional to the amount of ATP present. As cells multiply or as the amount of energy produced in a sample increases, the amount of ATP in a sample will increase. As cells lyse or as they become less active, the amount of ATP will decrease.

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The fluorescence test indicates the amount of intact and damaged cell membranes in a sample. Two stains are added to a sample containing bacteria and phage, which are absorbed by membranes of the intact and damaged cells.

When light is introduced to these two stains, they emit light at two different wavelengths, which is proportional to the amount of live and dead cells. When bacteria become infected, the cell membrane may change before the cell lyses

(Guttman et al. 2005) and the amount of each wavelength of light emitted may increase or decrease.

The purpose of using these three different tests to detect phage infection patterns is to determine if one more clearly shows phage infection. Also, one test may show signs of infection sooner in time than other tests, which would make it more desirable to use.

2.9 Literature Review Summary

Sludge bulking is a common problem encountered in activated sludge systems, and causes reduced settleability of biological flocs. Traditional methods used to control sludge bulking are time consuming and costly. Biocontrol of sludge bulking with phages is a novel idea for controlling sludge bulking.

Phages have been successfully isolated from activated sludge and used against bacteria for biocontrol. One study showed one such phage was successfully reduced sludge bulking induced by a filamentous bacteria (Kotay et al. 2011).

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The plate method is a common way to isolate phages. This method of detection does not work well for filamentous bacteria, so methods of detection will be explored in this thesis, which are absorbance, bioluminescence, and fluorescence tests. Phage infection patterns may be better detected or seen earlier in time with one test over the others, which will make it a more desirable method to detect phages.

When a phage of S. natans is isolated from activated sludge using these new methods, it is important that it is a virulent phage that immediately induces the lytic cycle. This will allow for immediate lysis of S. natans, which will quickly reduce sludge bulking induced by this bacteria type.

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CHAPTER III

MATERIALS AND METHODS

3.1 Materials

This chapter presents the materials used throughout this research thesis.

Tables 3.1 through 3.3 list the materials, equipment, and chemicals that are referred to throughout this chapter.

Table 3.1: Materials Material Description Manufacturer Location Sterile polypropylene 50 50 mL Centrifuge Tubes mL disposable centrifuge Fisher Scientific Fair Lawn, NJ tubes Sterile polypropylene 15 Corning 15 mL Centrifuge Tubes mL centrifuge tube with Corning, NY Incorporated CentriStar™ Cap Sterile polypropylene L- L-shaped Cell Spreader Fisher Scientific Fair Lawn, NJ shaped cell spreader 1, 2, 5, 10, and 25 mL Disposable serological Fisher Scientific Fair Law, NJ Pipets sterile pipets 200 µL and 1000 µL Hamburg, Autoclavable Eppendorf AG epT.I.PS. Pipet Tips Germany 10-100 µL and 100-1000 Hamburg, Automatic Pipets Eppendorf AG µL capacity Germany 0.5-10 mL capacity Hamburg, Repeat Pipet Eppendorf AG Repeater® plus Germany Combitips Plus 5 mL, Hamburg, Repeat Pipet Tips Eppendorf AG sterile Germany Sterile 1.5 mL clear Microcentrifuge Tubes polypropylene Fisher Scientific Fair Lawn, NJ microcentrifuge tubes

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Table 3.1 Continued Material Description Manufacturer Location 1, 2, 4 and 8 oz Bottles Polypropylene bottles Fisher Scientific Fair Lawn, NJ 10mL (12 mL) sterile Tuttlingen, 10 mL syringe polypropylene NORM- Henke Sass Wolf Germany JECT™ Luer Lock syringe Sterile 0.20 µm SFCE Corning 0.20 µm Filter Corning, NY membrane filter Incorporated Pechiney Plastic Laboratory Film Parafilm M Menasha, WI Packaging Corning 125 mL Baffled Flask 125 mL baffled glass flask Corning, NY Incorporated Corning 50 mL Baffled Flask 50 mL baffled glass flask Corning, NY Incorporated Orange polypropylene 25 Corning Orange Cap Corning, NY mm cap Incorporated Identi-Plugs®, expanded Jaece Industries, North Tonawanda, Small Foam Plugs polyether foam, Inc. NY autoclavable 0.45 µm cellulose acetate low protein binding Corning Filter Sterilized Water Corning, NY membrane, polystyrene Incorporated sterile container Sterile 12 well clear Becton Dickinson 12 Well Plate polypropylene Franklin Lakes, NJ Labware Multiwell™ plate Sterile assay plate, with Corning Clear 96 Well Plate low evaporation lid, flat Corning, NY Incorporated bottom, polystyrene Assay plate, no lid, flat Corning White 96 Well Plate bottom, non-sterile, Corning, NY Incorporated polystyrene Assay plate, no lid, flat Corning Black 96 Well Plate bottom, non-sterile, Corning, NY Incorporated polystyrene

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Table 3.2: Equipment Equipment Description Manufacturer Location Denver Digital Balance Read to 0.0000 g Instruments Arvada, CO Compnay International Centrifuge Centra CL2 Model # 120 Equipment Company

Orbital shaker table with Shaker Table adjustable speeds and Lab-Line durations

Odyssey Hach Loveland, CO Spectrophotometer Company Synergy™ 4 Multi- BioTek® Detection Microplate Plate reader Instruments, Winooski, VT Reader Inc. Gen5™ Microplate Data BioTek® Collection & Analysis Plate reader software Instruments, Winooski, VT Software Inc. Statistical analysis IBM SPSS Software Somers, NY software Corporation Omni Homogenizer TH Homogenizer Marietta, GA International Vortex Mixer with Fisher Vortex Fair Lawn, NJ Millipore Bedford, MA adjustable speeds Scientific

Reverse Osmosis Water Elix 10 Millipore Bedford, MA

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Table 3.3: Chemical Chemical Manufacturer Location BacTiter-Glo™ Reagent Promega Corporation Madison, WI

LIVE/DEAD® BacLight™ Invitrogen™ Eugene, OR Stain

Glycerol higly purified MP Biomedicals, LLC Solon, OH

Basingstoke, SR0105B Yeast Oxoid Ltd Hampshire, Autolysate Supplement England

Agar, for biochemistry, Acros Organics Geel, Belgium powder

Becton, Dickinson and Difco™ Nutrient Broth Sparks, MD Company

Fisher Scientific Sodium chloride Fair Lawn, NJ Company

Fisher Scientific Glycine Fair Lawn, NJ Company

Fisher Scientific Chloroform Fair Lawn, NJ Company

3.1.1 Bacteria and Bacteriophages

Bacteria used for propagation, enrichment, and titration of bacteriophages were Escherichia coli ATCC® 11303™ and Sphaerotilus natans ATCC® 15291 from the American Type Culture Collection (Manassas, Va.). The bacteriophage

T2 ATCC® 11303-B2™ was from the American Type Culture Collection

(Manassas, Va.) was used against E. coli ATCC® 11303™ in absorbance, bioluminescence, and fluorescence tests. Other unidentified bacteriophages used in absorbance, bioluminescence, and fluorescence tests and spot tests

20 were isolated from activated sludge processes at wastewater treatment plants.

The procedure for isolating phages from activated sludge can be seen in section

3.2.4 of this chapter. A summary of the samples collected can be seen in Table

4.1 in the Chapter 4.

3.1.2 Media

ATCC Medium #1103 Broth: Sphaerotilus CYGA Medium was used to grow S. natans contained 2.5 g Bacto™ casitone, 5.0 g glycerol highly purified, and 1 vial SR0105B Yeast Autolysate Supplement for every 500 mL of reverse osmosis water. Agar, for biochemistry, powder was added at 1.5% for hard agar or 0.5% for soft agar. The media was autoclaved for 20 minutes at 121°C.

BD233000 Nutrient Broth + 0.5% NaCl was used to grow E. coli and contained 8.0 g of Difco™ Nutrient Broth and 0.5% NaCl for every 1 L of reverse osmosis water. Agar powder was added at 1.5% for hard agar or 0.5% for soft agar. The media was autoclaved for 20 minutes at 121°C.

3.2 Methods

3.2.1 Quantification of Cell Density

McFarland Equivalence Turbidity Standards (Remel, Lenexa, Ks.) were used to estimate the density of bacteria present in samples. The absorbance at

590 nm [abs(590 nm)] was taken on the Odyssey spectrophotometer for each

McFarland Equivalence Turbidity Standard (0.5, 1.0, 2.0, 3.0, and 4.0) in a 1 cm x 1 cm methacrylate cuvette, which corresponds to a cell density (x 108/mL), and

21 plotted, as seen in Figure 1. These readings were only performed one time. In the future, the absorbance McFarland Standards should be taken and a new figure should be generated every time cell density is determined. This will ensure accuracy of the results.

Cell Density (x108/mL) 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0.9

0.8 0.7 y = 0.2029x + 0.0019 0.6 R² = 0.9998 0.5 0.4 0.3

0.2 Absorbance(590nm) 0.1 0 0.0 1.0 2.0 3.0 4.0 5.0 McFarland Equivalence Turbidity Standards

Figure 3.1: McFarland Equivalence Turbidity Standards Used to Estimate Cell Density

For bacteria cultures, the abs(590 nm) was taken and the equation obtained from the trendline in Figure 1 was used to find the estimated density of cells: y=0.2029x+0.0019 where y is the abs(590 nm) of the sample and x is the cell density (x108/mL). If the absorbance for E. coli was used, the units would be reported as x108 E. coli/mL.

3.2.2 Propagation and Harvesting of T2 Phage

T2 phage was propagated by the double layer agar plate method. In this method, 50 mL of Nutrient broth (NB) + 0.5% NaCl was inoculated with E. coli from a streak plate and incubated at 37°C overnight. A stock of E. coli

22 bacteriophage T2 with a titer of 109 pfu/mL was diluted to concentrations of 107,

105, and 103 pfu/mL using NB + 0.5% NaCl. Soft agar was prepared and kept in a water bath at 45°C. In a sterile test tube, 10 µL of each dilution was mixed separately into 0.5 mL of the overnight E. coli culture then 10 mL of soft nutrient agar was added to each tube. Triplicates were made of each dilution. Each test tube’s contents were gently swirled and poured over a prepared double layer hard agar plate pre-warmed to 37°C, allowed to harden, and incubated overnight at 37°C inverted. The next day, the top layer of soft agar was scraped into 50 mL centrifuge tubes using an L-shaped cell spreader and rinsed with 3 mL of NB +

0.5% NaCl. Then, 10 mL of NB + 0.5% NaCl was added to each tube. The soft agar layer and diluent were then centrifuged for 20 minutes at 2500 rpm. The top liquid was removed from the centrifuge tubes with a sterile pipet and dispersed in

1 mL aliquots into microcentrifuge tubes. The microcentrifuge tubes were placed in a labeled holder and stored at 4°C protected from light.

3.2.3 Titer of T2 Phage

The titer of T2 phage was determined using the plate method. Nutrient broth (NB) 0.5% NaCl was inoculated with E. coli from a streak plate and incubated at 37°C overnight. A stock of T2 phage was diluted with NB + 0.5%

NaCl to 10-2, 10-3, 10-4, 10-5, and 10-6. Soft agar was prepared and kept in a water bath at 45°C. For each phage dilution, 100 µL of each was mixed separately with 0.5 mL of the overnight E. coli culture and 5 mL of soft nutrient agar in a 15 mL centrifuge tube. Triplicates of each dilution were prepared.

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Each soft agar mixture was poured over a pre-warmed hard agar plate, allowed to harden, and incubated overnight at 37°C inverted. The next day, the plaques on each plate were counted using the whole plate count method. Plates that contained less than 30 plaques or greater than 300 plaques were discarded. The titer was calculated according to the following equation:

For example:

3.2.4 Collection and Filtration of Wastewater Samples for Isolation of Phages

Samples were collected from the activated sludge process of wastewater treatment plants in sterile 1L or 8 oz bottles. The samples were labeled with the collection site and date. The samples were named by taking the first four letters of the treatment facility’s location and the collection time. For example the second time a sample was collected from the City of Wapakoneta, the sample was named “Wapa2.” The samples were stored on ice during transportation to the lab. Once in the lab, the samples were immediately processed. The samples were allowed to settle, then 30 mL of the supernatant was collected from each sample and placed into a 50 mL centrifuge tubes. The samples were centrifuged at 1000 rpm for 10 minutes. From each tube 10 mL of the top liquid was collected into a 10 mL syringe then filtered through a 0.20 µm filter into a sterile 2 oz (about 60 mL) bottle. The filter was rinsed with 1 mL of sterile 0.25 M

24 glycine into the bottle. The bottles were labeled with the original sample name followed by “phage filtrate” and the date processed and stored at 4°C.

Next, 30 mL of the settled sludge on the bottom of the sample collected from the activate sludge was pipetted into a 50 mL centrifuge tube centrifuged for

10 minutes at 1000 rpm. Approximately half of the top liquid was poured off and replaced with 20% glycerol and mixed. The sample was labeled with the original sample name followed by “SASC” for “settled activated sludge concentrate” and the date the sample was processed. The samples were stored at -20°C.

3.2.5 Growth of Sphaerotilus natans

A hard agar CYGA plate was streaked with S. natans using a sterile swab.

The plate was inverted and incubated at 30°C for two days then wrapped with laboratory film. The plate was stored at room temperature because when the plates were stored at -4°C, the bacteria died within two days.

Using a sterile loop, 10 to 20 mL of sterile CYGA media in a baffled 125 mL flask was inoculated with S. natans from the streak plate and covered with a sterile orange cap that allowed oxygen exchange. The flask was secured to a shaker table and shook continuously at room temperature at 100 rpm to promote oxygen exchange.

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3.2.6 Preparation of 12 Well Multiwell™ Plate for Use in Absorbance,

Bioluminescence, and Fluorescence Tests for E. coli and T2 Phage

A culture of E. coli was prepared by inoculating 20 mL of sterile Nutrient

Broth (NB) + 0.5% NaCl with E. coli from a streak plate and incubated overnight at 37°C. In order to estimate the cell density of the sample, the absorbance at

590 nm was taken in a 1 cm x 1 cm methacrylate cuvette on the Odyssey

Spectrophotometer that was first zeroed on sterile NB + 0.5% NaCl. Dilutions of

107, 106, and 105 of E. coli were prepared using sterile NB + 0.5% NaCl in a 12 well Multiwell™ plate. The plate was gently shaken to uniformly distribute the bacteria in the wells. A stock of T2 phage with a titer of 109 pfu/mL was diluted to

104 pfu/mL and 103 pfu/mL using sterile NB + 0.5% NaCl in microcentrifuge tubes then distributed to the same Multiwell™ plate. The 12 well plate was stored at room temperature, 30°C, or 37°C between taking samples for testing. Figure 3.2 shows the layout of the 12 well plate.

Column Column Column Column 1 2 3 4 105 E. 105 E. 105 E. coli/mL coli/mL coli Row 1 103 102 pfu/mL pfu/mL no phage

106 E. 106 E. 106 E. coli/mL coli/mL coli Row 2 103 102 pfu/mL pfu/mL no phage

107 E. 107 E. 107 E. coli/mL coli/mL coli Row 3 103 102 pfu/mL pfu/mL no phage

Figure 3.2 : Layout of the 12 Well Plate for E. coli and T2 Phage

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3.2.7 Preparation of 12 Well Multiwell™ Plate Used in Absorbance,

Bioluminescence, and Fluorescence Test for E. coli and E. coli Phage

Enrichment

Approximately 20 mL of sterile Nutrient Broth (NB) + 0.5% NaCl was placed in a approximately 25 mL glass bottle with a metal screw on cap and was inoculated with E. coli from a streak plate and incubated overnight at 37°C. The absorbance at 590 nm was taken in a 1 cm x 1 cm methacrylate cuvette on the

Odyssey Spectrophotometer that was first zeroed on sterile NB + 0.5% NaCl.

The cell density was estimated from this absorbance. From this absorbance, the cell density was calculated from the McFarland Standard described above. This overnight E. coli culture was diluted to concentrations of 107, 106, and 105 E. coli/mL using sterile NB + 0.5% NaCl in a 12 well plate.

. “Troy2 E. coli Enrichment 1” was used as the phage enrichment, which is described in section 3.2.12 below. This phage enrichment was used because the presence of coliphages was confirmed through a spot test on a lawn of E. coli. After phages of E. coli were evidenced, the titer was assumed to be 109 pfu/mL (Costa et al. 2008), which was confirmed through a titer. The original

“Troy2 E. coli Enrichment 1 sample was diluted to 105, 104, and 103 pfu/mL using

NB + 0.5% NaCl in microcentrifuge tubes. These dilutions were added to the E. coli dilutions already in the 12 well plate described above. The layout of the plate can be seen in Figure 3.3. The difference between this set up and the set up for

E. coli and T2 phage is the additional samples containing 104 pfu/mL. Plates were prepared in this manner for the desired number of readings, one plate for

27 each time point reading. The plates were stored in an incubator room at 37°C when not in use.

Column Column Column Column 1 2 3 4 105 E. 105 E. 105 E. 105 E. coli/mL coli/mL coli/mL coli/mL Row 1 104 103 102 pfu/mL pfu/mL pfu/mL no phage

106 E. 106 E. 106 E. 106 E. coli/mL coli/mL coli/mL coli/mL Row 2 104 103 102 pfu/mL pfu/mL pfu/mL no phage

107 E. 107 E. 107 E. 107 E. coli/mL coli/mL coli/mL coli/mL Row 3 104 103 102 pfu/mL pfu/mL pfu/mL no phage

Figure 3.3: Layout of the 12 Well Multiwell™ Plate for E. coli and E. coli Phage Enrichment

3.2.8 Preparation of 12 Well Multiwell™ Plate Used in Absorbance,

Bioluminescence, and Fluorescence Test for S. natans and S. natans Phage

Enrichment

In a sterile baffled 125 mL flask, 10 to 15 mL of sterile CYGA was inoculated with S. natans from a streak plate and allowed to incubate for 2 to 9 days. The S. natans culture was homogenized with a sterile soft tissue probe, then the absorbance at 590 nm was taken in a 1 cm x 1 cm methacrylate cuvette on the Odyssey Spectrophotometer that was first zeroed on sterile CYGA. The cell density was estimated with the McFarland Standards previously described

28 using this absorbance. Dilutions of 107, 106, and 105 S. natans/mL were prepared using sterile CYGA media in a 12 well plate.

“Troy2 S. natans Enrichment 1 P1” was used as the phage enrichment, which is described in the section 3.2.11 below. This sample was diluted to 10-1,

10-2, and 10-3 pfu/mL using CYGA in microcentrifuge tubes. These dilutions were then distributed to the 12 well plate so that it contained the dilutions as seen in

Figure 3.4 below. The difference between the plate set up of this experiment and previous set ups is that the concentration of phage in the samples is unknown.

Column Column Column Column 1 2 3 4 106 S. 105 S. 105 S. 105 S. natans natans natans natans Row 1 10-2 10-3 10-4 pfu/mL pfu/mL pfu/mL no phage

106 S. 106 S. 106 S. 106 S. natans natans natans natans Row 2 10-2 10-3 10-4 pfu/mL pfu/mL pfu/mL no phage

107 S. 107 S. 107 S. 107 S. natans natans natans natans Row 3 10-2 10-3 10-4 pfu/mL pfu/mL pfu/mL no phage

Figure 3.4: Layout of the 12 Well Multiwell™ Plate for S. natans and S. natans Phage Enrichment

3.2.9 Absorbance Test of Phage Infection

Absorbance tests were performed using two different machines: the

Odyssey spectrophotometer and the Synergy™ 4 machine. The Odyssey was used to take initial absorbance readings of bacteria cultures that were to be used

29 in absorbance, bioluminescence, and fluorescence tests performed on the

Synergy™ 4 machine. The absorbance was set to 590 nm and zeroed on about

3 mL sterile growth media in a 1 cm x 1 cm methacrylate cuvette. The abs(590 nm) of about 3 mL of the culture contained in a 1 cm x 1 cm methacrylate cuvette was then read. The absorbance value was compared to the McFarland

Standards graph in Figure 3.1 in order to estimate the cell density of the culture.

For the absorbance test using E. coli and T2 phage, a clear 96 well plate was prepared using the same dilutions seen in Figure 3.2, and for absorbance tests using E. coli and an E. coli phage enrichment, the dilutions in Figure 3.3 were used. For each plate, the first row and first column were left empty. Each dilution (100 µL each) was pipetted into the appropriate well and a 100 µL blank of NB + 0.5% NaCl included. Single samples or triplicates of each dilution were prepared. The Biotek Synergy™ 4 machine was set up to read the absorbance at 590 nm and 630 nm using Gen5™ Software, and the protocol can be seen in

Appendix A. This procedure was performed every hour for 5 to 8 hours, leaving

1 row empty between each new setup.

Since S. natans grows in clumps, the samples for absorbance tests with homogenized S. natans and an S. natans phage enrichment were distributed to clear 96 well plate from the 12 well plate at time 0 and this plate was used for each time point during the tests, and the plate was protected from light between uses. The dilutions used in the 96 well plate can be seen in Figure 3.4. For each dilution, 100 µL was pipetted into the appropriate well and a 100 µL blank of

CYGA was included, and the first row and first column were left empty.

30

Triplicates of each dilution were prepared, leaving 1 row empty between each setup. The BioTek Synergy™ 4 machine was set up to read the absorbance at

590 nm and 630 nm using Gen5™ Software, and the protocol can be seen in

Appendix A.

3.2.10 BacTiter-Glo™ Microbial Cell Viability Assay

The BacTiter-Glo™ Microbial Cell Viability Assay (Promega Corporation,

Madison Wi.) was used in the bioluminescence tests. This method determines the amount of functioning cells in a sample by quantifying the amount of ATP present. The BacTiter-Glo™ Reagent contains a lysing detergent that lyses the cells in the sample in order to release the ATP in the cells and a thermostable luciferase (Ultra-Glo™ Recombinant Luciferase) that produces light during a

2+ reaction in the presence of Mg , O2, and ATP. The light emitted, proportional to the amount of ATP in the sample, was detected by the Synergy™ 4 machine.

3.2.10.1 Preparation of BacTiter-Glo™ Reagent

The BacTiter-Glo™ Reagent consisted of 2 components that were stored at -20°C: the BacTiter-Glo™ Substrate and the BacTiter-Glo™ Buffer. These two components were allowed to thaw and equilibrate to room temperature, then the buffer was added to the substrate and gently mixed by inverting until the substrate was dissolved, which took less than one minute. The reagent was then distributed into 1 mL aliquots in sterile 1.5 mL clear polypropylene microcentrifuge tubes ((Fisher Scientific, Fair Lawn, N.J.) or poured into a sterile

31 polypropylene 15 mL centrifuge tube with CentriStar™ Cap (Corning

Incorporated, Corning, N.Y.) and wrapped in aluminum foil or paper towels to protect from light. If the reagent was not used within eight hours, it was stored at

4°C for up to four days, -20°C for up to one week, or -70°C for up to one month.

3.2.10.2 Bioluminescence (ATP) Test

For the bioluminescence test involving E. coli and T2 phage, a white 96 well plate was prepared using the same dilutions seen Figure 3.2. Figure 3.3 was used for tests involving E. coli and E. coli phage enrichment. The first row and first column were left empty for each plate. For each dilution, 100 µL was pipetted into the appropriate well, as well as a 100 µL blank of NB + 0.5% NaCl.

BacTiter-Glo™ Reagent (Promega Corporation, Madison Wi) was prepared according to the manufacturer’s instructions. BacTiter-Glo™ Reagent was added in equal volumes of reagent to sample (100 µL for this case) to each filled well.

Single samples or triplicates of each dilution were prepared. The Biotek

Synergy™ 4 machine was set up to read the luminescence of each sample using Gen5™ Software, and the protocol can be seen in Appendix A. This procedure was performed every hour for 5 to 8 hours, leaving 1 row empty between each new setup.

For bioluminescence tests with S. natans and an S. natans phage enrichment, at time 0, 96 well plates were made for each time point from a prepared 12 well plate using the same dilutions seen in Figure 3.4. For each dilution, 100 µL was pipetted into the appropriate well and a 100 µL blank of

32

CYGA was included, and the first row and first column were left empty.

Triplicates of each dilution were prepared, leaving 1 row empty between each setup. These plates were stored protected from light. The Synergy™ 4 machine was set up to read the luminescence of each sample using Gen5™ Software, and the protocol can be seen in Appendix A. For each sample, 100 μL of

BacTiter-Glo™ Reagent was added just before testing.

3.2.11 L7012 LIVE/DEAD® BacLight™ Bacterial Viability Kit

The LIVE/DEAD® BacLight™ Bacterial Viability Kit (Invitrogen™, Eugene,

Or.) was used in the fluorescence tests. This method detects the amount of intact (“live”) and damaged (“dead”) cells present in a sample. It consists of two stains: SYTO® 9 green-fluorescent nucleic acid stain, which binds with both intact and damaged membranes, and propidium iodide red-fluorescent nucleic acid stain, which only binds with damaged membranes and causes a reduction in the green fluorescence. Ultimately, live bacteria with intact membranes will be stained green and dead bacteria or bacteria with damaged membranes will be stained red. The excitation/emission wavelengths for the SYTO® are around

480/500 nm while the excitation/emission wavelengths for propidium iodide are around 490/635 nm. The Synergy™ 4 machine was set up to emit an excitation wavelength of 485 nm and to detect emission wavelengths of 530 nm and 630 nm using Gen5™ software.

33

3.2.11.1 Preparation of L7012 LIVE/DEAD® BacLight™ Stain

The two components of this stain were stored at -20°C protected from light. The stains were allowed to thaw to room temperature before opening.

Three µL of SYTO® 9 and 3 µL of propidium iodide were combined and diluted with 1 mL of filter sterilized water. For every 6 µL of combined volume, 1 mL of filter sterilized water was used to dilute the stains. The diluted stain was wrapped in aluminum foil or paper towels and protected from light. The stain was kept at room temperature and used within eight hours of combining or discarded.

3.2.11.2 Fluorescence (Membrane) Test

Plates for the fluorescence test involving E. coli and T2 phage were set up with the same dilutions seen in Figure 3.2, while tests involving E. coli and a phage source used dilutions seen in Figure 3.3. The first row and first column were left empty on each plate. One hundred µL of each dilution was pipetted into the appropriate well, as well as a 100 µL blank of NB + 0.5% NaCl. Live/Dead

Stain was prepared according to the manufacturer’s instructions and added in equal volumes of stain to sample (100 µL for this case) to each filled well. Single samples or triplicates of each dilution were prepared. The Biotek Synergy™ 4 machine was set up to read the fluorescence of each sample using Gen5™

Software, and the protocol can be seen in Appendix A. This procedure was performed every hour for 5 to 8 hours, leaving 1 row empty between each new setup.

34

For fluorescence tests involving S. natans and an S. natans phage enrichment, 96 well plates were made for each time point from a prepared 12 well plate using the same dilutions seen in Figure 3.4 at the beginning of the experiment. For each dilution, 100 µL was pipetted into the appropriate well and a 100 µL blank of CYGA was included. The first row and first column were left empty on each plate. Triplicates of each dilution were prepared, leaving 1 row empty between each setup. These plates were stored protected from light. The

Synergy™ 4 machine was set up to read the fluorescence of each sample using

Gen5™ Software, and the protocol can be seen in Appendix A. The live/dead stain was added just before testing in100 µL volumes for each sample.

3.2.12 Enrichment of Phage Filtrate with S. natans

The purpose of enriching the phage filtrate with S. natans is to amplify the number of any phages of S. natans that may be present in the sample. Sterile

CYGA media (8 to 10 mL) in a sterile 125 mL baffled flask was inoculated with S. natans from a streak plate, covered with an orange cap, and incubated at room temperature on a shaker table that was shaking continuously at 100 rpm for 2 days or until thick growth was noticed. The S. natans culture was with a black sterile soft tissue probe for at least 30 seconds at half speed. Longer homogenization may be necessary for thick cultures. Bacteria should appear to be evenly distributed throughout the media. In a 50 mL baffled flask, 500 µL of

10X concentrated CYGA and 500 µL of the homogenized S. natans and 5 mL of phage filtrate were combined, sealed with a foam plug, and gently swirled. The

35 flask was wrapped in a paper towel or placed in a divided box in order to protect the phages from light and placed on the shaker table continuously shaking at 100 rpm and allowed to incubate for 2 to 3 days, or until growth was noticed in the flask. The flask was vortexed for 5 to 10 seconds and the contents were transferred to a sterile 30 to 50 mL glass beaker after flaming the neck of the flask. The contents of the beaker were suctioned into a 10mL syringe then filtered through a 0.20 µm filter into a sterile 2 oz bottle that was labeled with original sample name followed by “S. natans Enrichment #,” where # represented the number of times the sample had been enriched, and the date the enrichment was filtered. The phage enrichment was stored at 4°C.

3.2.13 Enrichment of Phage Filtrate with E. coli

Phage filtrates were enriched with E. coli in order to increase the number of any phages of E. coli that may have been present. A sterile flask containing

20 mL of sterile Nutrient Broth + 0.5% NaCl was inoculated with E. coli from a streak plate and incubated overnight at 37°C. In a sterile glass bottle, 500 µL of sterile 10X NB + 0.5% NaCl, 500 µL of the overnight E. coli culture, and 5 mL of phage filtrate was combined and gently swirled. The bottle was then incubated overnight at 37°C, protected from light. The next day, this phage enrichment was collected in a 10 mL sterile syringe and filtered through a 0.2 µm filter. The filtered phage enrichment was stored at 4°C.

36

3.2.14 Spot Test of Phage Enrichment on S. natans Lawn

A lawn of S. natans was prepared by streaking a CYGA hard agar plate in five directions. This produced a lawn of S. natans that covered the entire plate.

Three circles were drawn on the outside of the streak plate where the phage enrichment was to be spotted, as shown in Figure 3.5.

Figure 3.5: Layout of Spot Test

Ten µL of phage enrichment was placed on the S. natans lawn in the middle of each circle. A control plate with no phage enrichment was prepared.

The plates were inverted and incubated at 30°C for 3 days. Any plaques were collected. If the plate did not have any plaques, it was placed in a plastic bag and stored at 4°C.

37

3.2.15 Collection of Phage Plaques and Phages from Spot Test of Phage

Enrichment on S. natans

Each plaque was labeled on the outside of the hard agar plate with P# (P1 for plaque 1, for example). A 15 mL centrifuge tube was labeled with collection site and number, enrichment and number, and P# (Troy2 S. natans Enrichment 1

P1, for example). Each plaque was cut out using the end of a L-shaped cell spreader and placed in a labeled centrifuge tube, and a new cell spreader was used for each plaque. into each centrifuge tube containing a plaque, 5 mL of sterile CYGA media was placed and gently mixed. Each centrifuge tube was wrapped in paper towels to protect any phages from light stored at 4°C.

Under a fume hood, 3 drops of chloroform were added to each centrifuge tube using a sterile pipet. The tubes were gently swirled, wrapped in paper towels, and left at room temperature overnight. The next day, the tubes were vortexed for 5 to 10 seconds, and then the contents of the centrifuge tubes were transferred to sterile 50 mL beakers covered in aluminum foil. The liquid was suctioned into a 10mL then filtered through a 0.20 µm filter into a sterile 2 oz bottle that was labeled with collection site and number, enrichment and number, and P#. The bottles were stored at 4°C protected from light.

3.2.16 Titration of Filtered and Non-filtered T2 Phage

Since activated sludge samples were filtered in order to obtain phage filtrates, it was necessary to determine if the titer of the phage filtrate would be reduced as a result of filtering.

38

Fifty mL of sterile Nutrient Broth (NB) + 0.5% NaCl was inoculated with E. coli from a streak plate and incubated overnight at 37°C. Phage dilutions of 10-6,

10-7, and 10-8 pfu/mL were prepared in microcentrifuge tubes by diluting a stock of T2 phage using sterile NB + 0.5% NaCl.

To filter the T2 phage, the plunger of a 10mL syringe was removed and

100 µL of the same T2 phage stock as above was added to the syringe along with 900 µL of sterile NB + 0.5% NaCl. The plunger was replace and the contents were filtered through a 0.20 µm filter into a microcentrifuge tube. Phage dilutions of 10-6, 10-7, and 10-8 pfu/mL were prepared from this filtrate in microcentrifuge tubes.

Soft agar was prepared and kept in a water bath at 45°C. Triplicates were prepared of the soft agar over lay by adding 5 mL of soft nutrient agar, 500 µL of the overnight E. coli culture, then 100 µL of the appropriate T2 phage dilution to a

15 mL centrifuge tube. The tubes were gently swirled then poured over hard agar double layer plates pre-warmed to 37°C. After the top agar hardened, the plates were inverted and incubated at 37°C overnight.

The next day, the plaques on each plate were counted using the whole plate count method. Plates that contained less than 30 plaques or greater than

300 plaques were discarded. The titer was calculated according to the following equation:

39

The results of this titer were compared to the results of the titer of the same stock that was not filtered. A statistical analysis was performed on these two data sets using SPSS Software to see if the reduction due to filtering was significant.

3.2.17 Titration of T2 Phage Introduced to Hard Agar

Fifty mL of sterile Nutrient Broth (NB) + 0.5% NaCl was inoculated with E. coli from a streak plate and incubated overnight at 37°C. A stock of T2 phage with a titer of 109 pfu/mL was diluted in triplicates to 104 pfu/mL using sterile NB +

0.5% NaCl in microcentrifuge tubes. Twenty mL of hard nutrient agar and 75 mL of soft nutrient agar was prepared, autoclaved, and kept in a water bath at 60°C.

Five mL of hard agar was placed into three 50 mL centrifuge tubes, then 500 µL of the 104 pfu/mL sample of T2 phage was added to each tube (one tube contained one replicate sample). The tubes were mixed by swirling and allowed to harden. After hardening, 5 mL of sterile NB + 0.5% NaCl was added to each tube and the tubes were homogenized with a sterile black soft tissue probe until the agar was dissolved. The tubes were then for 20 minutes at 2500 rpm. The top liquid from each tube was collected separately and placed in a 15 mL centrifuge tube and stored at 4°C. From each tube, 10-5, 10-6, and 10-7 dilutions were prepared in microcentrifuge tubes. From the T2 phage stock used above,

10-5, 10-6, and 10-7 dilutions were prepared in microcentrifuge tubes. Five hundred µL of the overnight E. coli culture was added to twelve 15 mL centrifuge tube. One hundred µL of the appropriate phage dilution was added to each tube,

40 followed by 5 mL of soft nutrient agar, then the tubes were swirled to mix. Each tube was poured over a hard agar double layer plate that was labeled and pre- warmed to 37°C. After the top agar hardened, the plates were inverted and placed in a 37°C incubator and incubated overnight.

The next day, the plaques on each plate were counted using the whole plate count method. Plates that contained less than 30 plaques or greater than

300 plaques were discarded. The titer was calculated according to the following equation:

The titer of the T2 phage that was retrieved from hard agar was compared to the titer of the same stock of T2 phage that was not introduced to hard agar in order to see if the titer was reduced by the hard agar.

3.2.18 Growth Curve of S. natans using Absorbance

S. natans was inoculated into fresh CYGA broth from a culture in CYGA broth. Three or seven days later, this culture was homogenized for 30 seconds and 100 µL was pipetted into six different wells on a clear 96 well plate and the remaining empty wells were filled with 200 µL of sterile water. The Synergy™ 4 machine was set to 30°C and to continuously shake. The absorbance of each at

590 nm and 630 nm was taken every 30 minutes for 24 hours using both the normal read and sweep read modes. The exact procedure can be seen in

Appendix A.

41

3.2.19 Absorbance Test on E. coli and BacTiter-Glo ™Reagent Kept at Room

Temperature

It was hypothesized that since the BacTiter-Glo™ reagent contains a lysing reagent, taking samples from bioluminescence test that showed evidence of phage infection and enriching the phage in these samples in a fresh bacteria culture may not be possible. This experiment tested whether or not the lysing reagent would become deactivated after being kept at room temperature for different amounts of time.

BacTiter-Glo Reagent was prepared on Day 0. This stock solution was divided into 10 micro centrifuge tubes that contained 1 mL each. On Day 0, 2 of these aliquots were left at room temperature, protected from light. The remaining aliquots were placed in the freezer, protected from light. On Day 1, 2 of the aliquots were removed from the freezer and left at room temperature, protected from light. This procedure was performed on Days 2, 3, and 4. On Day 3, an E. coli culture was inoculated in Nutrient Broth + 0.5% NaCl.

On Day 4, the 108 cells/mL overnight culture of E. coli was diluted to 107 cells/mL. A 96 well plate was prepared as seen below in Figure 3.5. The wells that contain more than one component have 100 µL of each component. The wells that contain one component have 200 µL of that component. The red font represents blank samples.

42

E. coli + E. coli + Day 0 E. coli + Day 1 E. coli + Day 2 E. coli + Day 3 Sterile Water E. coli + E. coli + Day 0 E. coli + Day 1 E. coli + Day 2 E. coli + Day 3 Sterile Water E. coli + E. coli + Day 0 E. coli + Day 1 E. coli + Day 2 E. coli + Day 3 Sterile Water

Sterile Water + E. coli + Day 4 NB + Day 4 NB Sterile Water Day 4 Sterile Water + E. coli + Day 4 NB + Day 4 NB Sterile Water Day 4 Sterile Water + E. coli + Day 4 NB + Day 4 NB Sterile Water Day 4 Figure 3.6: Plate Layout for E. coli and BacTiter-Glo Assay Absorbance Tests

The 96 well plate was placed in the Synergy™ 4 machine. The temperature was set to 37°C. The machine took an absorbance reading at 590 nm every 30 minutes. The plate was shaken for 10 seconds before each reading. The protocol followed by the Gen5 Software can be found in Appendix

A.

3.2.20 E. coli and BacTiter-Glo Reagent™ Dilutions Absorbance Tests

As mentioned above, the BacTiter-Glo™ reagent contains a lysing detergent that may make enrichment of phages from samples that contain the reagent difficult. This experiment tested whether or not the lysing detergent could be diluted enough so it would not interact with all the bacteria in a fresh culture.

BacTiter-Glo™ was prepared then diluted with filter-sterilized water. The dilutions were 100, 10-1, 10-2, …, 10-10. One hundred µL of E. coli and 100 µL of the appropriate dilution were placed in a well of a clear 96-well plate. Blanks of

200 µL were also included for each dilution. Triplicates were made of each

43 sample. The E. coli placed in the wells had a concentration of 1x108 cells/mL.

The absorbance was taken of each sample every 30 minutes for 24 hours in the

Synergy™ 4 machine. The incubator in the machine was set to 37°C. The protocol performed by the Gen5 Software can be seen in Appendix A.

3.2.21 Absorbance Tests of Mixed Microbial Community with Different Diluents

A sample was collected from the City of Dayton Activated Sludge tank on

December 22, 2010. This sample was allowed to settle, and a portion of the supernatant was collected. This supernatant was diluted to concentrations of

1:102, 1:104, and 1:106 supernatant: diluent. The diluents in this case were filter- sterilized water, nutrient broth + 0.5% NaCl, or CYGA. For each sample, 100 μL was place in a clear 96 well plate, with triplicates of each sample. Blanks of sterile water, nutrient broth + 0.5% NaCl, and CYGA were placed in the same plate in triplicates. The Synergy™ 4 machine was set to 30°C and absorbance readings at 590 nm were taken every 30 minutes for 24 hours. Appendix A shows the protocol followed by the Gen5 Software for this experiment.

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CHAPTER IV

RESULTS AND DISCUSSION

The goal of this thesis is to identify patterns associated with phage infection using the absorbance, bioluminescence, and fluorescence broth tests.

The model bacteria and phage was E. coli and T2 phage. Once patterns of phage infection were identified for each broth test using this model, the applicability of these patterns to E. coli and unidentified phages of E. coli isolated from activated sludge was determined. These same tests were also performed on S. natans and possible phages of S. natans isolated from activated sludge in order to determine whether or not phage infection could be identified by the same patterns.

4.1 Absorbance Test Results for E. coli and T2 Phage

The Absorbance test detects the amount of light that is blocked by particles in the material through which the light is traveling. In this case, E. coli cells block the light in this test, and the amount of light blocked will increase as the cells multiply. As cells lyse, the amount of light that can pass through the sample will increase, which will cause the absorbance value to decrease. It is hypothesized that this test will indicate phage infection in the following way: growth of E. coli noted by an increase in absorbance, infection of E. coli by T2

45 phage showed by a stable absorbance reading in samples that contain phage as the control continues to increase and death of E. coli due to cell lysis indicated a decreasing absorbance in samples that contain phage while the control continues to grow. The results show the effects of different concentrations of phage on different concentrations of E. coli.

Upon inspection of Figure 4.1 below, samples that contain T2 phage increase in absorbance for the first 3 hours, remain around this absorbance for 2 hours, and then decrease in absorbance. These phases can be interpreted as growth of E. coli, stationary phase, and dominant cell lysis due to infection of the

E. coli by the T2 phage. The stationary phase may be caused by growth and death rates, due to phage infection, being about the same, or phages inhibiting bacteria reproduction. The control sample has continued increase in absorbance for the duration of the test period until the last time point, indicating a typical growth curve. In the case of the control, the decrease in absorbance in the last time point is due to normal cell death, not infection by phage. The bacteria in the samples that contain phage begin to die after 3 hours, rather than continue to multiply as in the control, due to infection and inhibition or lysis via phage.

Figure 4.2 shows a slightly different pattern after the first 2 hours. The control increases rapidly for the duration of the test, while the samples that contain phage increase very slowly and very little. This slow increase can be attributed to the growth and death rate (due to cell lysis caused by phage infection) being about the same, or by growth being hindered as the phage DNA takes over the cell. The endpoints of the curves should also be noted. The

46 control has a much higher absorbance at the end of the time trial, compared to the samples that contain phage. This same pattern can be observed in Figures

4.3 through 4.7 as well. Because this pattern is observed in all the absorbance tests, it is an acceptable model for detecting phage infection.

In addition, the samples that contain 103 pfu/mL generally have a lower absorbance than the samples that contain 102 pfu/mL. This is due to the higher ratio of phage to bacteria in the 103 pfu/mL samples, so there are more phages to infect the bacteria, which results in more bacteria being lysed and a lower absorbance.

A comparison of ratios of E. coli to T2 phage can be seen in Figure 4.8.

When bacteria are present in concentrations much higher than phage, the infection of the bacteria by phage is less evident than with lower concentrations.

To clarify, the higher ratios increase in absorbance faster than the lower ratios.

In lower ratios of bacteria to phage, the phage appears to be more effective at controlling the growth of bacteria, shown by the small increase in absorbance, compared to higher ratios. For this reason, the recommended ratio of bacteria to phage for detecting phage in environmental samples is 1000:1 or 100:1. The

1000:1 bacteria to phage ratio was also used by other researchers to detect phages from activated sludge samples (Kotay et al. 2011; Viazis et al. 2011).

While the ratio of bacteria to phage is important, the concentrations of the bacteria and the phage also appear to have importance in detecting phage infection. In Figure 4.9, a comparison is made of the 1000:1 ratio of bacteria to phage that contain 106 E. coli/mL with 103 pfu/mL and 105 E. coli/mL and 102

47 pfu/mL of T2 phage. Even though the same ratio of bacteria to phage is used, the sample that was prepared using the higher concentration of bacteria has a higher absorbance at each time point than the lower concentration. The same is true for Figure 4.10, which shows a similar comparison for the 10000:1 ratio.

From these results, it can be concluded that with environmental samples, lower concentrations of bacteria may work better because the expected titer of the phage in the samples obtained from activated sludge is low – around 103 pfu/mL to 105 pfu/mL for E. coli (Tanji et al. 2003) and is expected to be lower for slow growing and less abundant microorganisms (Kotay et al. 2011; Withey et al.

2005), such as filamentous bacteria. The lower concentrations of bacteria may allow for clearer detection of phage infection because the absorbance will be lower.

One disadvantage of using lower concentration of bacteria is the delayed growth of the control, as seen in Figures 4.2 and 4.5. The control does not show a major increase in absorbance until 3 to 4 hours after the test began, compared to 1 to 2 hours for higher concentrations of E. coli. This avoidance of taking unnecessary readings that do not give any information about phage infection can be accomplished by waiting 3 to 4 hours after combining E. coli and phage dilutions to take any absorbance readings on the samples. This will allow a baseline control growth curve to be established so that the samples that contain phage can be compared to it. When using other types of bacteria, a growth curve of just bacteria should be obtained in order to determine when to begin sampling with that specific species.

48

It should also be noted that in general, as the concentration of E. coli increases, the separation between the 103 pfu/mL, 102 pfu/mL, and control curves increases. For low concentrations of E. coli (105 cells/mL), the lack of separation may be caused by the low ratio of bacteria to phage, along with the relatively low concentration of bacteria. The phages may be infecting so many cells that growth (an increase in absorbance) is not apparent in samples that contain phage. As seen in Figures 4.8 and 4.9, the lower the concentration of bacteria, the lower the absorbance compared to higher concentrations.

Preliminary absorbance tests were performed using only one sample for each bacteria/phage dilution, as seen in Figure 4.1. Absorbance tests performed later in research were performed using three samples of each bacteria/phage dilution and the average absorbance of the three values for each dilution was plotted, as seen in Figures 4.2 through 4.7. The error bars in these figures represent the standard deviation.

In addition to the number of samples, the incubation temperatures were different for initial experiments. In initial tests, the Multiwell™ plates containing the bacteria/phage dilutions were stored at room temperature between testing.

Because of the low temperature, there was little growth in the most of the control samples in most cases, as indicated by a low range of absorbance values in most cases, and emphasizes the need to have a well established baseline for the control. These results do not yield indications of growth or phage infection and are therefore not included in this section, but the data can be found in Appendix

B. One experiment performed under these conditions yielded growth in the

49 control sample, as well as the samples that contained phage, as seen in Figure

4.1.

In later tests (Figures 4.2 through 4.7), the Multiwell™ plates containing the bacteria/phage dilutions were stored in an incubator at 37°C between test times. Since bacteria have a higher growth rate at higher temperatures, the absorbance range for these experiments were generally larger than the ones performed with samples incubated at lower temperatures (Figure 4.1), so growth and phage infection are evident.

For future tests performed on a bacteria and a phage source that possibly contains phage, the pattern observed in Figure 4.2 should be investigated.

When bacteria are infected by phage, the absorbance should increase very slowly as compared to the control sample. If this occurs, the slow increase can be attributed to phages infecting the bacteria and inhibiting it from multiplying or causing it to lyse.

107 E. coli/mL

0.12

0.1 No Phage 0.08

10^2 PFU/mL Abs (590 (590 Abs nm) 0.06 10^3 PFU/mL 0.04 0 2 4 6 8 Time (hrs)

Figure 4.1: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at Room Temperature

50

105 E. coli/mL

0.090

0.080 0.070 No Phage 0.060 10^2 PFU/mL 0.050

Abs (590 (590 Abs nm)(AU) 10^3 PFU/mL 0.040 0 2 4 6 8 Time (hrs)

Figure 4.2: Absorbance Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

106 E. coli/mL

0.100

0.080 No Phage 0.060 10^2 PFU/mL

Abs (590 (590 Abs nm)(AU) 0.040 10^3 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.3: Absorbance Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

107 E. coli/mL

0.120 0.100 0.080 No Phage 0.060 10^2 PFU/mL

Abs (590 (590 Abs nm)(AU) 0.040 10^3 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.4: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

51

105 E. coli/mL

0.120

0.100

0.080 No Phage

0.060 10^2 PFU/mL

Abs(590 Abs(590 nm)(AU) 10^3 PFU/mL 0.040 0 2 4 6 8 Time (hrs)

Figure 4.5: Absorbance Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

106 E. coli/mL

0.140 0.120 0.100 No Phage 0.080 10^2 PFU/mL 0.060 Abs(590 Abs(590 nm)(AU) 0.040 10^3 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.6: Absorbance Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

107 E. coli/mL

0.140 0.120 0.100 No Phage 0.080 10^2 PFU/mL 0.060 Abs(590 Abs(590 nm)(AU) 0.040 10^3 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.7: Absorbance Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

52

Ratios of Bacteria to Phage

0.090

0.080 100000:1 E. coli: phage 0.070 10000:1 E. coli: phage 0.060 1000:1 E. coli: phage

0.050 Abs(590 Abs(590 nm)(AU)

0.040 100:1 E. coli: phage 0 2 4 6 8 Time (hrs)

Figure 4.8: Comparisons of Ratios of E. coli to Phage

1000:1 E. coli to Phage 0.075 0.070 0.065 0.060 10^6 E. coli, 10^3 0.055 phage 0.050 10^5 E. coli, 10^2

Abs (590 (590 Abs nm)(AU) 0.045 phage 0.040 0 2 4 6 8 Time (hrs)

Figure 4.9: Comparison of the 1000:1 E. coli: Phage Ratio

10000:1 E. coli to Phage

0.090 0.080

0.070 10^7 E. coli, 10^3 0.060 Phage 0.050 10^6 E. coli, 10^2 Abs (590 (590 Abs nm)(AU) 0.040 Phage 0 2 4 6 8 Time (hrs)

Figure 4.10: Comparison of the 10000:1 E. coli: Phage Ratio

53

4.2 Bioluminescence (ATP) Test Results for E. coli and T2 Phage

The Bioluminescence test detects the amount of ATP, or cell energy, present in a sample. All cells produce ATP in order to function. More cell activity results in more ATP production, so the amount of ATP in the sample is indicative of the amount of viable bacteria cells or the amount of cell activity. As seen in the Figures 4.11 through 4.16 below, for example, the higher the concentration of

E. coli, the higher the luminescence reading. When a cell becomes infected, the amount of ATP produced inside the cell may change before the cell lyses due to infection by phage. It was hypothesized that by measuring the amount of ATP produced over time, infection of E. coli may be able to be detected sooner than with the absorbance or live/dead test.

As seen in Figure 4.12, an increase in luminescence occurs in samples that contain T2 phage sooner than the control. This increase is followed by a decrease in luminescence, indicating a decrease in ATP and cell activity. It is assumed that this decrease in ATP occurs due to bacteria lysis induced by phage infection. A possible explanation for this rapid increase in ATP of the samples containing phage is that when the phage takes over the cell, more ATP is required to manufacture phage components than is needed in cells that are not infected (i.e. control). This increase in ATP in samples that contain phage before the control is observed in all of the figures in this section (4.11 through 4.16), so it is concluded to be a characteristic of phage infection.

The data from Figures 4.11 through 4.13 were normalized to better showcase this effect, and can be seen in Figures 4.17 through 4.19. As

54 described above, the ATP produced in the samples containing phage is initially much higher than the control, as seen in Figure 4.17 for example. The peak luminescence value is followed by a generally steady decrease in luminescence relative to the control. The endpoints of the phage samples are much lower than the control. These figures also show that the ratio of bacteria to phage as well as the concentration of bacteria do not affect the detectability of infection patterns in the ratios and concentrations that were tested, unlike with the absorbance test.

Because the samples that contain lower concentrations of bacteria exhibit a lower total change in luminescence, there may be a limit to the ratio of bacteria to phage that shows phage infection patterns.

Another pattern detected in Figure 4.12 is that samples which contain T2 phage increase in luminescence for the first portion of the test then remain around the same luminescence for the remainder of the test or decrease in luminescence. These phases can be interpreted as growth of E. coli, stationary phase where the growth and death rate about equal, and cell lyses in reduced activity, presumably due to infection of the E. coli by T2 phage. The control sample has a continued increase in luminescence for the duration of the test period, indicating continuous growth. The difference between the growth curves of the samples that contain phage and the growth curves of the controls is that the stationary phase occurs very early in the test period for phage samples, while it does not occur at all in the control. The stationary period occurs in this manner due to the phages infecting the cells and causing inhibition or lysis of bacteria within the sample. Also, the peak luminescence value and the endpoint

55 luminescence value are lower for the samples that contain phage than the control. These same patterns are common to almost all the figures in this section, so they can be used to indicate phage infection.

In addition, the samples that contain 103 pfu/mL have an initial higher luminescence increase than the samples that contain 102 pfu/mL, and the peak luminescence is also lower and occurs sooner, as in Figure 4.16 for example. It is hypothesized that the luminescence increases faster for the sample containing a phage concentration of 103 pfu/mL than the sample containing 102 pfu/mL due to the higher ratio of phages to bacteria. Since there are more phages to infect the bacteria, it is assumed that more ATP is produced in preparation to manufacture phage components. The peak for the 103 pfu/mL concentration of phage is lower because more bacteria in these samples are lysed before the bacteria in the 102 pfu/mL sample due to the higher ratio of phages to bacteria.

This pattern is seen in Figures 4.11 through 4.16 below. For environmental samples, a higher initial of increase in ATP should be watched for in samples that contain higher concentrations of phage.

Experiments presented in this section were performed using three samples of each bacteria/phage dilution and the average luminescence of the three values was plotted for each dilution, as seen in Figures 4.11 through 4.16.

The error bars in these figures represent the standard deviation.

In addition to the number of samples, the incubation temperatures were different for initial experiments. The Multiwell™ plates containing the bacteria/phage dilutions were stored at room temperature or at 30°C between

56 testing. Most of these experiments resulted in little growth of the bacteria in the control sample due to the low temperature, as indicated by a low range of bioluminescence values in most cases. These results do not yield much information about growth and phage infection and are therefore not included in this section, but the data can be found in Appendix C. The remaining results are from experiments in which the Multiwell™ plates containing the samples were stored in 37°C incubators between test periods. The slow growth of the E. coli at lower temperatures indicates the need to have a well established base growth of

E. coli, as well as proper growth conditions.

When experiments are performed on bacteria and a phage source that may contain phages of the bacteria of interest in future tests, samples that contain phage should exhibit the same patterns as those seen in Figure 4.13.

The luminescence should initially increase faster than the control, but peak sooner and at a lower luminescence value. Also, for lower concentrations of phage, the initial increase in ATP should be lower than higher phage concentrations, but higher than the control. The peak luminescence reading should also occur sooner and lower than the control, but higher and later than higher phage concentrations. The endpoint values for samples containing phage should be lower than the control as well.

57

105 E. coli/mL

2000.0

1500.0

1000.0 No Phage 10^2 PFU/mL 500.0 10^3 PFU/mL Luminescence(RLU) 0.0 0 2 4 6 Time (hrs)

Figure 4.11: ATP Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

106 E. coli/mL

2500.0 2000.0 1500.0 No Phage 1000.0 10^2 PFU/mL 500.0 10^3 PFU/mL Luminescence(RLU) 0.0 0 2 4 6 Time (hrs)

Figure 4.12: ATP Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

107 E. coli/mL

12000.0 10000.0 8000.0 6000.0 No Phage 4000.0 10^2 PFU/mL 2000.0 10^3 PFU/mL Luminescence(RLU) 0.0 0 2 4 6 Time (hrs)

Figure 4.13: ATP Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

58

105 E. coli/mL 105 E. coli/mL

30000.0 600.0 500.0 10^3 20000.0 400.0 300.0 Phage 200.0 10000.0 10^2 100.0 Phage 0.0 0.0

0 2 4 No Luminescence(RLU) Luminescence(RLU) 0 2 4 6 Time (hrs) Phage Time (hrs)

(a) (b) Figure 4.14: (a) ATP Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 (b) Zoomed to Show Initial ATP Increase of Phage Samples

106 E. coli/mL 106 E. coli/mL

30000.0 2500.0 2000.0 10^3 20000.0 1500.0 Phage 1000.0 10^2 10000.0 500.0 Phage 0.0 No 0.0 Phage

0 2 Luminescence(RLU) Luminescence(RLU) 0 2 4 6 Time (hrs) Time (hrs)

(a) (b) Figure 4.15: (a) ATP Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2 (b) Zoomed to Show Initial ATP Increase of Phage Samples

107 E. coli/mL

30000.0 25000.0 20000.0 15000.0 10^3 Phage 10000.0 10^2 Phage 5000.0 No Phage

Luminescence(RLU) 0.0 0 1 2 3 4 5 6 Time (hrs)

Figure 4.16: ATP Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

59

105 E. coli/mL

2.000

1.500

1.000 Control 10^3 pfu/mL 0.500

10^2 pfu/mL Luminescence(RLU) 0.000 0 2 4 6 8 Time (hrs)

Figure 4.17: Normalized Data of Figure 4.11

106 E. coli/mL

2.500 2.000 1.500 Control 1.000 10^3 pfu/mL 0.500 10^2 pfu/mL Luminescence(RLU) 0.000 0 2 4 6 8 Time (hrs)

Figure 4.18: Normalized Data of Figure 4.12

107 E. coli/mL

3.500 3.000 2.500 2.000 Control 1.500 1.000 10^3 pfu/mL 0.500 10^2 pfu/mL Luminescence(RLU) 0.000 0 2 4 6 8 Time (hrs)

Figure 4.19: Normalized Data of Figure 4.13

60

4.3 Fluorescence (Live/Dead) Test Results for E. coli and T2 Phage

The Fluorescence test indicates the amount of intact (“live”) and damaged

(“dead”) membranes in the material being tested. For this test, a combination of two stains is added to a bacterial suspension. The membranes of all cells whether intact or damaged absorb one stain, and the membranes of the damaged cells absorb the other stain. When the dead cells absorb the dye, the

“live” dye is displaced. The two stains fluoresce at two different wavelengths when an excitation wavelength is applied, both of which are detected when the test is performed. The amount of light emitted from each type of stain is indicative of the quantity of intact live and damaged or dead cells. The hypothesis behind this test is that as the cells become infected, the membrane of the cell may change and absorb more of the dead stain before the cell lyses and dies, and the amount of each wavelength of light emitted may increase or decrease, and phage infection may be indicated sooner than with absorbance or bioluminescence by tracking these changes over time.

The pattern observed in Figure 4.20 below in the samples that contain phage is an increase in the ratio of live to dead cells for the first 2 hours, followed by a steady decrease in the ratio for the remainder of the test. These results indicate a growth phase then a death phase. Because the death phase begins after only 2 hours, it is not due to natural cell death, but lysis induced by phage infection. The curve for the control sample increases for the duration of the experiments, which is another indication that the samples that contain phage are dying due to phage infection. Many of the figures below exhibit this same pattern

61 of growth and death. In some figures, such as Figure 4.28, the samples that contain phage exhibit an increase (growth phase), leveling off (stationary phase), and decrease (death phase) in the ratio of live to dead cells, rather than a growth then immediate death phase. This occurs while the control sample continuously grows. This is shown by the samples that contain phage and indicates a pattern of phage infection, which is also seen in the bioluminescence and absorbance tests. The stationary phase that occurs after 2 hours is presumed to be caused by some cells being infected with phage and ultimately lysing or being inhibited from growing due to infection, while other cells’ membranes remain intact since they are not infected. The continued growth of the control is another indicator that the patterns shown by samples that contain phage are due to phage infection rather than natural cell death.

As with the absorbance tests, as the concentration of E. coli increases, the separation between the 103 pfu/mL, 102 pfu/mL, and control curves increases, as especially apparent in Figures 4.27, 4.28, and 4.29. For low concentrations of E. coli (105 cells/mL), the lack of separation may be caused by the high ratio of phage to bacteria, along with the relatively small number of cells. The phage may be taking over so many cells that growth (an increase in ratio) is not outstanding in samples that contain phage.

This observation brings to light the importance of the bacteria to phage ratio, as well as the concentration of bacteria, just as with the absorbance test.

When higher ratios of bacteria to phage are present, a higher growth rate is observed, and evidence of phage infection (a decline or a leveling off in the ratio

62 from the control) is shown at a higher ratio of live to dead cells. Figure 4.30 highlights this. With lower ratios of bacteria to phage, the change in ratio is smaller, but the pattern of phage infection described above is still detected.

For phage samples collected from activated sludge processes, titers are expected to be low. For this reason, it is desirable to use a lower concentration of bacteria, i.e. 105 E. coli/mL, for the fluorescence test. If a lower concentration of E. coli is used, the lower ratios are able to be achieved. If the titer of phage in the sample happens to be higher than expected, infection of bacteria will still be able to be observed by a low increase in the ratio of live to dead cells. However, if too high a concentration of bacteria is used, the infection of cells may go unnoticed due to the high amount of growth exhibited by uninfected cells.

Unlike with the absorbance test, where growth of the control was not observed until 3 to 4 hours after testing began, the live/dead test indicates growth of the control after 2 hours in samples that contain 105 E. coli/mL. If the fluorescence test is used, readings of samples may begin immediately after the bacteria and phage samples are prepared.

Preliminary fluorescence tests were performed using only one sample for each bacteria/phage dilution, as seen in Figures 4.20 through 4.23. Experiments performed later in research were performed using three samples of each bacteria/phage dilution and the average ratio of live to dead cells of the three values obtained for each dilution were plotted, as seen in Figures 4.24 through

4.29. The error bars in these figures represent the standard deviation.

63

In addition to the number of samples, the incubation temperatures were different for initial experiments. In preliminary experiments, the Multiwell™ plates containing the samples were incubated at 30°C. Some of these experiments resulted in little growth of the E. coli due to the low temperature, especially in samples containing 105 E. coli/mL, so these results were omitted from this section. Data for these experiments can be found in Appendix D. In the remaining experiments, the Multiwell™ plates containing the bacteria/phage dilutions were stored in an incubator at 37°C. The experiments in which the samples were incubated at 30°C resulted in a slower growth for the control samples

When testing a phage source against bacteria using the fluorescence test, the pattern that should be looked for is the ratio of live to dead cells for the samples that contain phage should begin to level off or decrease in absorbance as the control sample continues to increase, as observed in the figures below.

The leveling off or decrease in the ratio of phage samples as the control continues to increase is the presumed indication of phage infection using this test.

64

106 E. coli/mL

15.00

10.00 No Phage 10^2 PFU/mL 5.00

10^3 PFU/mL Ratio ofLive Dead to 0.00 0 1 2 3 4 5 Time (hrs)

Figure 4.20: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 30°C, Trial 1

107 E. coli/mL

20.00

15.00

10.00 No Phage 10^2 PFU/mL 5.00

10^3 PFU/mL Ratio ofLive Dead to 0.00 0 1 2 3 4 5 Time (hrs)

Figure 4.21: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 30°C, Trial 1

65

106 E. coli/mL

15.00

10.00 No Phage 10^2 PFU/mL 5.00

10^3 PFU/mL Ratio ofLive Dead to 0.00 0 1 2 3 4 5 6 Time (hrs)

Figure 4.22: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 30°C, Trial 2

107 E. coli/mL

20.00

15.00

10.00 No Phage 10^2 PFU/mL 5.00

10^3 PFU/mL Ratio ofLive Dead to 0.00 0 1 2 3 4 5 6 Time (hrs)

Figure 4.23: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 30°C, Trial 2

66

105 E. coli/mL

10.00

No Phage 5.00 10^2 Phage

0.00 10^3 Phage

Ratio ofLive Dead to 0 2 4 6 Time (hrs)

Figure 4.24: Live/Dead Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

106 E. coli/mL

12.00 10.00 8.00 6.00 No Phage 4.00 10^2 Phage 2.00 0.00 10^3 Phage Ratio ofLive Dead to 0 2 4 6 Time (hrs)

Figure 4.25: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

107 E. coli/mL

12.00 10.00 8.00 6.00 No phage 4.00 10^2 Phage 2.00 0.00 10^3 Phage Ratio ofLive Dead to 0 2 4 6 Time (hrs)

Figure 4.26: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 1

67

105 E. coli/mL

15.00

10.00 No Phage 5.00 10^2 PFU/mL

0.00 10^3 PFU/mL Ratio ofLive Dead to 0 2 4 6 8 Time (hrs)

Figure 4.27: Live/Dead Test for 105 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

106 E. coli/mL

15.00

10.00 No Phage 5.00 10^2 PFU/mL 0.00 10^3 PFU/mL

Ratio ofLive Dead to 0 2 4 6 8 Time (hrs)

Figure 4.28: Live/Dead Test for 106 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

107 E. coli/mL

15.00

10.00 No Phage 5.00 10^2 PFU/mL

0.00 10^3 PFU/mL Ratio ofLive Dead to 0 2 4 6 8 Time (hrs)

Figure 4.29: Live/Dead Test for 107 E. coli/mL and T2 Phage Stored at 37°C, Trial 2

68

Ratios of Bacteria to Phage 9.00

8.00 7.00 100000:1 E. coli: 6.00 phage 5.00 10000:1 E. coli to 4.00 phage 3.00 1000:1 E. coli: phage

2.00 Ratio ofLive Dead to 1.00 100:1 E. coli: phage 0.00 0 2 4 6 8 Time (hrs)

Figure 4.30: Comparisons of Different E. coli to T2 Phage Ratios for Live/Dead Test

4.4 Comparison of Absorbance, Bioluminescence, and Fluorescence Tests

for E. coli and T2 Phage

With all three test methods (absorbance, bioluminescence, and fluorescence), growth of E. coli as well as infection of E. coli by T2 phage can be detected, and distinct patterns for each test were observed. These patterns can be clues to phage infection induced by phages collected from activated sludge.

This is important because the presence of phages infective to select bacteria is unknown, so if the same patterns are observed, the incidence of these phages can be concluded.

With the absorbance test, infection is evidenced by a leveling off and/or a decrease in absorbance in samples that contain phage as the control continues to increase, which is indicative of cells lysing or being inhibited due to phage infection. Another indicator of phage infection using the absorbance test is a

69 very slow increase in absorbance compared to the control, which is assumed to be caused by phages inhibiting or lysing the bacteria. Also, the absorbance values for the phage samples will be lower than the control at the end of the experiment.

Evidence of phage infection may be detected sooner than the absorbance test with the bioluminescence (ATP) test because in the samples that contain phage, the luminescence increases faster than the control in the beginning of the test. It is assumed that this rapid increase is due to an increased production of

ATP induced by phage infection. Also, the peak luminescence reading is reached earlier in the test and is lower than the control sample. After the peak, the luminescence decreases in the samples that contain phage, presumably because phage infection causes the infected cells to lyse and die. As with the absorbance test, the final luminescence values observed at the end of the test period will be lower for samples that contain phage than the control. The repeatability of these patterns indicates that the luminescence is an excellent method for detecting phage infection.

In results from the fluorescence (live/dead) test, the samples that contain phage always show clear evidence of infection. In most cases, especially at higher concentrations of E. coli, the ratio of live to dead cells begins to increase slowly in the samples that contain phage than the control after 1 hour, which is an earlier indicator of phage infection than the absorbance test. In addition, the separation between the curves of the samples that contain phage is greater with the fluorescence test compared to the absorbance test. This shows that the

70 fluorescence test is more sensitive to changes in the samples that contain phage than the absorbance test. The absorbance test shows little increase and change in shape of the growth curves. The fluorescence test clearly shows an increase and eventual decrease in the ratio of live to dead cells, which is caused by phage infection. Just like with the absorbance and ATP tests, the endpoint live/dead ratio will be lower in samples that contain phage than the control.

4.5 Summary of Collected Samples of Activated Sludge

Table 4.1 below shows all the samples that were collected from activated sludge processes and filtered in order to isolate phage (called “phage filtrate”).

The table also shows which samples were enriched with S. natans or E. coli.

71

Table 4.1: Activated Sludge Samples and Enrichments S. natans E. coli Sample Location Filter Date Enrichment Enrichment Name Date Date City of Piqua, Ohio 12/17/2009 Piqu1 5/25/2010 6/15/2010 City of Troy, Ohio 12/17/2009 Troy1 5/25/2010 6/15/2010 City of Sidney, Ohio 12/22/2009 Sidn1 5/25/2010 6/15/2010 City of Dayton, 12/29/2009 Dayt1 5/25/2010 6/15/2010 Ohio Beverly Hills SD, Auglaize County, 12/29/2009 Beve1 5/25/2010 6/15/2010 Ohio Sherwood Forest SD, Auglaize 12/29/2009 Sher1 5/25/2010 6/15/2010 County, Ohio K/Z SD, Auglaize 12/29/2009 KZ1 5/25/2010 6/15/2010 County, Ohio Beverly Hills SD, Auglaize County, 5/10/2010 Beve2 5/25/2010 6/15/2010 Ohio Sherwood Forest SD, Auglaize 5/10/2010 Sher2 5/25/2010 6/15/2010 County, Ohio K/Z SD, Auglaize 5/10/2010 KZ2 5/25/2010 6/15/2010 County, Ohio City of Sidney, Ohio 5/10/2010 Sidn2 5/25/2010 6/15/2010 City of Troy, Ohio 5/10/2010 Troy2 5/25/2010 6/15/2010 City of 6/9/2010 Wapa1 6/30/2010 6/15/2010 Wapakoneta, Ohio City of Sidney, Ohio 6/9/2010 Sidn3 6/30/2010 6/15/2010 City of Piqua, Ohio 6/9/2010 Piqu2 6/30/2010 6/15/2010 City of 7/22/2010 Wapa2 Wapakoneta, Ohio City of Sidney, Ohio 7/22/2010 Sidn4 City of Troy, Ohio 7/22/2010 Troy3 City of Piqua, Ohio 9/8/2010 Piqu3 9/8/2010 City of Troy, Ohio 9/8/2010 Troy4 9/8/2010 City of Dayton, 11/16/2010 Dayt2 Ohio

72

4.6 Absorbance Test Results for E. coli and “Troy 2 E. coli Enrichment 1”

In this section, absorbance tests were performed using E. coli and phages of E. coli isolated from activated sludge. Similarities between infection patterns observed in these tests are compared to those observed with the E. coli and T2 phage model. In sections 4.7 and 4.8, bioluminescence and fluorescence tests are conducted using E. coli and the same phage source. A comparison of these three test methods is made in section 4.9.

The absorbance tests for E. coli and the “Troy 2 E. coli Enrichment 1” phage source and the absorbance tests for the model E. coli and T2 phage share similar patterns, as in Figure 4.31, for example. The absorbance for the samples that contain the phage source increase the same as the control for the first 3 hours of the experiment, indicating growth of E. coli, followed by a decrease in absorbance, indicating phage infection and lysing of E. coli, while the control sample continues to increase in absorbance, indicating continuous growth. Also, as with the experiments that involved E. coli and T2 phage, as the concentration of E. coli increases the separation between the growth curves increases, as seen in Figures 4.31 through 4.33. This is due to the high ratio of phages to bacteria.

The more phages there are, the more bacteria can be infected, and so more cells are lysed than in samples with lower concentrations of phage.

As seen in Figures 4.31 and 4.33 below, the samples that contain the E. coli phage enrichment “Troy 2 E. coli Enrichment 1” and 105 E. coli/mL increase in absorbance (grow) with the control for the first 3 to 4 hours. After this, the absorbance of the samples that contain phage generally decreases for the

73 remainder of the experiments, which is due to E. coli lysing or inhibition caused by phage infection, while the control sample continues to increase in absorbance.

This is similar to the pattern observed in the absorbance test using E. coli and T2 phage. As mentioned in the section on absorbance using E. coli and T2 phage, the control takes a longer time to show growth in samples that contain 105 E. coli/mL compared to samples that contain higher bacteria concentrations. To avoid taking unnecessary measurements in the future, readings should begin after 3 hours of mixing the bacteria with the phage source.

In Figures 4.32 and 4.35, which have E. coli concentrations of 106 E. coli/mL, and in Figures 4.33 and 4.36, which have E. coli concentrations of 107 E. coli/mL, the absorbance for all samples with phage increases the as control for the 1 to 2 hours. The samples that contain higher concentrations of phage decrease sooner and more rapidly than those that contain lower concentrations, which in expected because more phages are present to infect cells.

The similarities between this experiment and the E. coli and T2 phage model demonstrate that phages can be isolated from wastewater samples and phage infection can be tracked using the absorbance test when testing a phage source against E. coli.

74

105 E. coli/mL 0.140 0.120 0.100 0.080 No Phage 0.060 10^2 PFU/mL 0.040 10^3 PFU/mL

Abs(590 Abs(590 nm)(AU) 0.020 0.000 10^4 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.31: Absorbance Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1

106 E. coli/mL 0.140 0.120 0.100 0.080 No Phage 0.060 10^2 PFU/mL 0.040 10^3 PFU/mL

Abs(590 Abs(590 nm)(AU) 0.020 0.000 10^4 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.32: Absorbance Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1

107 E. coli/mL

0.150

0.100 No Phage 10^2 PFU/mL 0.050

10^3 PFU/mL Abs(590 Abs(590 nm)(AU) 0.000 10^4 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.33: Absorbance Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 1

75

105 E. coli/mL

0.150

0.100 No Phage 10^2 PFU/mL 0.050

10^3 PFU/mL Abs(590 Abs(590 nm)(AU) 0.000 10^4 PFU/mL 0 2 4 6 Time (hrs)

Figure 4.34: Absorbance Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2

105 E. coli/mL

0.150

0.100 No Phage 10^2 PFU/mL 0.050

10^3 PFU/mL Abs(590 Abs(590 nm)(AU) 0.000 10^4 PFU/mL 0 2 4 6 Time (hrs)

Figure 4.35: Absorbance Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2

107 E. coli/mL

0.200

0.150 No Phage 0.100 10^2 PFU/mL 0.050

10^3 PFU/mL Abs(590 Abs(590 nm)(AU) 0.000 10^4 PFU/mL 0 2 4 6 Time (hrs)

Figure 4.36: Absorbance Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source, Trial 2

76

4.7 Bioluminescence Test Results for E. coli and “Troy 2 E. coli Enrichment

1”

In Figures 4.37 through 4.39 below, the samples that contain 104 pfu/mL initially produce more ATP than any other phage concentration. This concentration reaches a peak luminescence reading sooner in time than lower concentrations. This peak is followed by a rapid decrease that continues for the remainder of the experiment. Just as with the E. coli and T2 phage model, this same pattern can be seen in subsequent lower phage concentrations. The 103 pfu/mL initially has higher luminescence readings than the 102 pfu/mL concentration, and the 102 pfu/mL concentration’s luminescence is higher than the control samples. After the highest peak in ATP release, the luminescence decreases rapidly, indicating a decreased production of ATP in the sample. This was attributed to the assumption that cells were lysing due to infection of the bacteria by phage, or that the ATP is being used for produce phage parts. Again, the increased creation of ATP by samples that contain phage higher than the control samples may be caused by the infected cells’ increased requirement for energy in order to manufacture new bacteriophages. Since this pattern was observed both here and in the E. coli and T2 phage model, it lends more evidence that this pattern is a sign of phage infection and should be looked for in tests on bacteria with an unknown phage source.

Another pattern that is the same in the E. coli and “Troy 2 E. coli

Enrichment1” phage source and the E. coli and T2 phage model is that the peak

ATP value is lower for higher concentrations of phage. In addition the ending

77 values of ATP were lower in all samples that contain phage than the control.

Because the same patterns with T2 phage were shown between with an unknown phage from activated sludge, the bioluminescence test is a viable way to track phage infection using E. coli.

78

105 E. coli/mL 105 E. coli/mL

30000.0 3000.0 2500.0 No 20000.0 2000.0 Phage 1500.0 10^2 1000.0 10000.0 500.0 PFU/mL 0.0 10^3 0.0 PFU/mL

0 5 10^4 Luminescence(RLU) Luminescence(RLU) 0 2 4 6 Time (hrs) PFU/mL Time (hrs)

(a) (b) Figure 4.37: (a) ATP Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage and (b) Zoomed View to Show Early Increase in ATP

106 E. coli/mL 106 E. coli/mL

5000.0 15000.0 No Phage 4000.0 3000.0 10000.0 10^2 2000.0 PFU/mL 5000.0 1000.0 10^3 0.0 0.0 PFU/mL

0 5 10^4 Luminescence(RLU) Luminescence(RLU) 0 2 4 6 8 Time (hrs) PFU/mL Time (hrs)

(a) (b) Figure 4.38: (a) ATP Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage and (b) Zoomed View to Show Early Increase in ATP

107 E. coli/mL

30000.0 25000.0 20000.0 No Phage 15000.0 10^2 PFU/mL 10000.0 5000.0 10^3 PFU/mL

Luminescence(RLU) 0.0 10^4 PFU/mL 0 2 4 6 8 Time (hrs)

Figure 4.39: ATP Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source

79

4.8 Fluorescence Test Results for E. coli and “Troy 2 E. coli Enrichment 1”

In Figure 4.40, all the concentrations of phage have about the same ratio of live to dead cells as the control for the first 2 hours of the test. The ratios begin to increase for each concentration except for 104 pfu/mL, which stays about the same during the experiment. The ratio for this sample is 10:1 bacteria to phage. This ratio of bacteria to phage may be too low to observe growth of the bacteria in the samples, but phage infection can be concluded since the control grows and this sample does not.

Also in Figure 4.40, The 103 pfu/mL concentration’s ratio of live to dead cells decreases after 4 hours. The ratio of phage to bacteria in this sample is

100:1, and this ratio allows infection of E. coli to be noted by the increase and decrease in ratio of live to dead cells, while the control continues to grow. The

102 pfu/mL (1000:1 ratio of bacteria to phage) and control’s ratio of live to dead cells increases during the entire test period, indicating continuous growth of bacteria in both samples. In the case of 102 pfu/mL, the concentration of this particular phage may be too low to cause a noticeable increase in the amount of dead bacteria cells.

Figure 4.41 shows growth of E. coli for all phage concentrations for the first hour of the experiment. The 104 pfu/mL concentration’s ratio of live to dead cells increases slower than the other concentrations then decreases for the remainder of the experiment (100:1 bacteria to phage ratio). The 103 pfu/mL concentration’s ratio increase through hour two, then decreases for the remainder of the experiment (1000:1 bacteria to phage ratio). The 102 pfu/mL

80 and control curves increase for the entire experiment (10000:1 bacteria to phage ratio). The increases and decreases in the live to dead ratios for the 100:1 and

1000:1 ratios of bacteria to phage indicate growth and decay, presumably due to lysis of bacteria cells in the samples, since the control exhibits growth during the entire experiment.

Figure 4.42 most clearly shows growth and death of the bacteria in samples that contain phage and it also shows the effects of the concentration of phage. Each curve shows growth in the first hour. The 104 pfu/mL concentration’s ratio rapidly declines after this initial growth phase and stays around the same level throughout the experiment (1000:1 bacteria to phage).

The 103 pfu/mL concentration’s ratio increases through hour 2, then progressively decreases for the remainder of the experiment (10000:1 bacteria to phage). The 102 pfu/mL concentration’s ratio follows the same pattern, but the decline takes more time (100000:1 bacteria to phage). The control sample increases in ratio throughout the experiment.

In this experiment, bacteria to phage ratio that repeatedly showed signs of phage infection was the 100:1. The 1000:1 ratio did not show patterns associated with infection in the 105 E. coli concentration sample, but it did in higher concentrations. This difference highlights the importance of not only testing different ratios of bacteria to phage, but also testing different concentrations of bacteria for the same ratio of bacteria to phage.

The E. coli and T2 phage model shows similar patterns to those presented in the E. coli and “Troy 2 E. coli Enrichment 1” fluorescence experiments. These

81 similarities attest that phages can be isolated from wastewater samples and phage infection can be tracked using the fluorescence test.

82

105 E. coli/mL

15.00

10.00 No Phage 10^2 PFU/mL 5.00 10^3 PFU/mL 0.00 10^4 PFU/mL

0 2 4 6 Ratio of 530 : nmnm 630 Time (hrs)

Figure 4.40: Live/Dead Test for 105 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source

106 E. coli/mL

15.00

10.00 No Phage 10^2 PFU/mL 5.00 10^3 PFU/mL 0.00 10^4 PFU/mL

0 2 4 6 Ratio of 530 : nmnm 630 Time (hrs)

Figure 4.41: Live/Dead Test for 106 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source

107 E. coli/mL

15.00

10.00 No Phage

5.00 10^2 PFU/mL 10^3 PFU/mL 0.00 10^4 PFU/mL 0 2 4 6

Ratio of 530 : nmnm 630 Time (hrs)

Figure 4.42: Live/Dead Test for 107 E. coli/mL and “Troy 2 E. coli Enrichment 1” Phage Source

83

4.9 Summary of Broth Tests Involving E. coli and Phage Source

With the absorbance tests conducted with E coli and a phage source, similar patterns to those observed with T2 phage model were shown in samples that contained unknown phages from activated sludge, as described in section

4.6. One difference between the infection patterns of T2 phage and the phage source is that the absorbance increases to a higher value before decreasing in tests using the phage source, especially at higher concentrations of bacteria.

The reason for this may be due to the phages infecting the E. coli more slowly than the T2 phage. If tests are not conducted for a long enough period, phage infection may go unnoticed when using high concentrations of bacteria. This highlights the importance of using low bacteria to phage ratios, as well as using a low concentration of bacteria to prepare these ratios.

Another challenge with the absorbance test is that using lower concentrations of bacteria (i.e. 105 cfu/mL) to prepare test samples, which was recommended in section 4.1, leads to a longer lag phase at the beginning of the experiment. Because of this, growth is not exhibited until 2 to 3 hours after reading begin. In future tests involving E. coli, readings should begin approximately 3 hours after mixing the bacteria and phages together.

The bioluminescence tests performed with E. coli and phages isolated from activated sludge demonstrated the same patterns shown by the T2 phage.

One possible challenge with using the bioluminescence test is that using lower concentrations of bacteria to prepare samples results in a lower overall change in luminescence values. Though the values are lower, the same changes are

84 observed, regardless of bacteria to phage ratio. This characteristic makes this test ideal because phages can be detected at high bacteria to phage ratios.

Though overall results were similar between the T2 phage model and the phage source for the fluorescence tests, the same ratios of bacteria to phage yielded inconsistent results. The ratio of 1000:1 bacteria to phage did not show phage infection when the samples was prepared using 105 cfu/mL (colony- forming-units per milliliter) bacteria concentration, but did show phage infection patterns when 106 cfu/mL was used to make the sample; therefore it is recommended to use multiple concentrations of bacteria to prepare the same bacteria to phage ratio.

As with the absorbance test, the phage source experiments yielded more growth of E. coli than the T2 phage experiments, as indicated by a larger increase in the live/dead ratio before a decrease occurs. Again, if experiments are not performed long enough, phage infection may be falsely unconcluded.

Regardless of which test method is used, a baseline growth curve for the chosen bacteria should first be established using the desired measurement method. This will reduce the number of necessary readings, save time, and save money by using less stain in the ATP and live/dead tests.

4.10 Growth of S. natans

Before the absorbance, bioluminescence, or fluorescence tests could be performed on S. natans, a reliable method to grow it had to be developed. It was determined that in order to obtain thick growth on a streak plate, a sterile swab

85 should be used rather than a sterile loop. Incubation at 30°C yielded more growth in comparison to incubation at room temperature and 37°C.

In order to grow S. natans in CYGA broth, oxygen exchange must be allowed. This was performed by inoculating broth in a baffled flask covered with a lid that allowed air to be exchanged, and was further promoted by incubating the culture on an orbital shaker table. The incubation of S. natans in broth was performed at room temperature, but incubating at 30°C may yield faster growth, as was shown by faster growth of S. natans on streak plates incubated at 30°C.

4.11 Tracking the Growth Curve of S. natans Using Absorbance

As seen in Figures 4.43 and 4.44, the absorbance test yields highly variable results for S. natans, as indicated by the high standard deviation. This is due to the morphology of the bacteria, which grows in sheaths and clumps together. The non-homogeneous growth pattern does not allow the bacteria to be evenly dispersed throughout the sample and causes light to be blocked in an uneven manner. For this reason, two different modes of absorbance were compared, the sweep read and the normal read. As shown in Figures 4.43 and

4.44, these two read methods yielded nearly the same results, which are both highly variable, and the standard deviation increase with time. Because of this high variability, the absorbance test is not recommended to track the growth and phage infection of S. natans by phage from a phage source.

Information that can be gathered from Figure 4.43 below is that S. natans begins to increase in growth after 5 hours. This growth continues up to 10 hours,

86 where the stationary phase is reached. For this reason, when testing samples for the presence of phage against S. natans, reading should begin 5 hours after the phage source is introduced to S. natans.

S. natans Growth Curve (Sweep Read)

1.000

0.800

0.600

0.400

Abs(590 Abs(590 nm)(AU) 0.200

0.000 0 5 10 15 20 25 Time (hrs)

Figure 4.43: S. natans Growth Curve Using Sweep Read Mode

S. natans Growth Curve (Normal Read) 1.000 0.900

0.800 0.700 0.600 0.500 0.400 0.300 Abs(590 Abs(590 nm)(AU) 0.200 0.100 0.000 0 5 10 15 20 25 Time (hrs)

Figure 4.44: S. natans Growth Curve Using Normal Read Mode

87

4.12 Absorbance Test Results for S. natans and “Troy 2 S. natans

Enrichment 1 P1”

As discussed section 4.11 above, the absorbance test yields highly variable results for S. natans due to its morphology. In the figures below, the samples that contain the phage source below do not exhibit the same growth, stationary, and death phases observed in experiments using E. coli and a phage source. These samples that contain the phage source follow the same pattern as the controls rather than decreasing as the control increases in absorbance.

Because the control does not exhibit a dramatic growth phase, it is likely that S. natans did not have enough time to grow in the control or in the phage samples, so even if phages were present, their effects on S. natans could not be seen.

88

105 S. natans/mL 0.060

0.055 No Phage 0.050 10^-4 Phage Dilution

Abs (590 (590 Abs nm) 0.045 10^-3 Phage Dilution 0.040 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.45: Absorbance Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

106 S. natans/mL 0.090

0.080 0.070 No Phage 0.060 10^-4 Phage Dilution

Abs (590 (590 Abs nm) 0.050 10^-3 Phage Dilution 0.040 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.46: Absorbance Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

107 S. natans/mL 0.180

0.160 0.140 0.120 No Phage 0.100 10^-4 Phage Dilution 0.080 Abs (590 (590 Abs nm) 10^-3 Phage Dilution 0.060 0.040 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.47: Absorbance Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

89

105 S. natans/mL

0.070

0.065 0.060 No Phage 0.055 10^-4 Phage Dilution 0.050

0.045 10^-3 Phage Dilution Abs(590 Abs(590 nm)(AU) 0.040 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.48: Absorbance Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

106 S. natans/mL

0.130

0.110 0.090 No Phage 0.070 10^-4 Phage Dilution

0.050 10^-3 Phage Dilution Abs(590 Abs(590 nm)(AU) 0.030 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.49: Absorbance Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

107 S. natans/mL

0.300 0.250 0.200 No Phage 0.150 10^-4 Phage Dilution

0.100 10^-3 Phage Dilution Abs(590 Abs(590 nm)(AU) 0.050 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.50: Absorbance Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

90

4.13 Bioluminescence Test Results for S. natans and “Troy 2 S. natans

Enrichment 1 P1”

In Figures 4.51 through 4.53 below, the samples that contain the phage source follow the same growth pattern as the control, implying that phage infection is not occurring in these samples. These figures show that all the samples tend to increase in luminescence for the duration of the experiments, indicating growth. The samples that contain the phage source, however, do not increase rapidly at the beginning of the test, as in experiments performed with E. coli. Figure 4.53 shows an increase in luminescence for the first 4 hours, followed by a decline from hour 4 to hour 6. Since all the samples, even the control, follow this pattern, the decline can be attributed to natural cell death rather than phage infection.

The high variability in the bioluminescence test, shown by the high standard deviations, can be attributed to the morphology of S. natans. The culture was homogenized before the experiments were performed, but the cells may not have been evenly distributed throughout the broth when the aliquots were distributed to the 96 well test plates for the experiment, so each aliquot may not have received the same amount of bacteria.

91

105 S. natans/mL

800.0

700.0 No Phage 600.0 10^-4 Phage Dilution 500.0 10^-3 Phage Dilution

Luminescence(RLU) 400.0 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.51: ATP Test for Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source

106 S. natans/mL

5400.0 4400.0 3400.0 No Phage 2400.0 10^-4 Phage Dilution 1400.0 10^-3 Phage Dilution

Luminescence(RLU) 400.0 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.52: ATP Test for Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source

107 S. natans/mL

6400.0 5400.0 4400.0 No Phage 3400.0 10^-4 Phage Dilution 2400.0 1400.0 10^-3 Phage Dilution

Luminescence(RLU) 400.0 10^-2 Phage Dilution 0 2 4 6 8 Time (hrs)

Figure 4.53: ATP Test for Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source

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4.14 Fluorescence Test Results for S. natans and “Troy 2 S. natans

Enrichment 1 P1”

In the figures below, the samples that contain phage exhibit the same pattern as the control samples for each experiment. In some cases, such as in

Figure 4.54, it appears that samples that contain phage may decrease in the live to dead ratio as the control increases, but the high standard deviation must be taken into consideration. The error bars overlap for the control and samples that contain phage, so these Figures are not reliable for indicating whether or not phages of S. natans are actually present.

In addition, the live to dead ratios changed very little throughout the experiments performed on S. natans and the phage source, unlike those performed on E. coli and T2 phage model. The S. natans experiments’ controls ratio of live to dead cells increased or decreased by about 0.2 during the experiments, while the E. coli and T2 control ratios increased between 7.0 and

11.0. This lack of growth in the S. natans during these experiments may be due to its slow growth rate, and emphasizes the need to have an established baseline of growth in the control samples before readings are taken.

As in the bioluminescence test, this high variability can be attributed to the morphology of S. natans and the bacteria not being distributed evenly in each aliquot.

93

105 S. natans/mL

1.70 1.60 1.50 No Phage 1.40 1.30 10^-4 Phage Dilution 1.20 10^-3 Phage Dilution 1.10 10^-2 Phage Dilution 0 2 4 6 8 Ratio of 530 : nmnm 630 Time (hrs)

Figure 4.54: Live/Dead Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

106 S. natans/mL

1.40

1.30 No Phage 1.20 10^-4 Phage Dilution 1.10 10^-3 Phage Dilution 1.00 10^-2 Phage Dilution

Ratio of 530 : nmnm 630 0 2 4 6 8 Time (hrs)

Figure 4.55: Live/Dead Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

107 S. natans/mL

1.60 1.40

1.20 No Phage 1.00 10^-4 Phage Dilution 0.80 10^-3 Phage Dilution 0.60 10^-2 Phage Dilution

Ratio of 530 : nmnm 630 0 2 4 6 8 Time (hrs)

Figure 4.56: Live/Dead Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 1

94

105 S. natans/mL

1.40

1.35 1.30 No Phage 1.25 10^-4 Phage Dilution 1.20

1.15 10^-3 Phage Dilution Ratio of530:630 1.10 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.57: Live/Dead Test for 105 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

106 S. natans/mL 1.60 1.50 1.40 No Phage 1.30 10^-4 Phage Dilution 1.20 10^-3 Phage Dilution Ratio of530:630 1.10 1.00 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.58: Live/Dead Test for 106 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

107 S. natans/mL

3.00

2.50 No Phage 2.00 10^-4 Phage Dilution

1.50 10^-3 Phage Dilution Ratio of530:630 1.00 10^-2 Phage Dilution 0 2 4 6 8 10 Time (hrs)

Figure 4.59: Live/Dead Test for 107 S. natans/mL and “Troy 2 S. natans Enrichment 1 P1” Phage Source, Trial 2

95

4.15 Summary of Broth Tests Involving S. natans and Phage Source

All three broth tests performed with S. natans and the phage source did not show the phage infection patterns that were detected with E. coli. The main hypothesis for the lack of evidence is that because of the slow-growing nature of

S. natans, not enough time was allowed for growth to be detected in neither the controls nor the phage samples. Readings were taken beginning immediately after S. natans was mixed with the phage source. An absorbance test was performed on S. natans to obtain a baseline growth curve (shown in section

4.11). This showed that S. natans begins to grow in the log phase 5 hours after the test began. In future tests performed with S. natans and a phage source, readings should begin 5 hours after mixing the bacteria and phage source.

Another challenge with using S. natans is the high variation in the samples. When S. natans was homogenized, the clumps of bacteria were broken up, but instead of staying homogenously distributed throughout the sample volume, the particles tended to settle to the bottom. Because of this, it is likely that each aliquot of did not receive exactly the same amount of S. natans.

In the future, homogenizing the culture for a longer period of time is recommended, as well as mixing the culture well before distributing the bacteria to aliquots.

The absorbance test yielded results with much larger variations than the bioluminescence and fluorescence tests. This is suspected to be due to the distribution of the S. natans throughout the sample. As S. natans grows, it tends to cluster together, which can give variable results for the same amount of bacteria when using the absorbance test. These preliminary tests suggest that

96 the absorbance test is not a good method to use for S. natans, but after the above recommendations have been implemented, more tests should be performed to confirm this hypothesis.

4.16 E. coli and BacTiter-Glo™ Reagent Absorbance Test

Because the BacTiter-Glo™ Reagent contains a lysing detergent that lyses the bacteria cells to release the ATP into the sample so it can be detected, the question was raised whether or not phages could be retrieved from samples that show phage infection and enriched in a fresh bacterial culture without lysing the bacteria. To test this, aliquots of the reagent were left at room temperature for 0 to 96 hours, protected from light, to see if the lysing detergent would become inactive within this time frame.

Figure 4.60 shows a control of E. coli and sterile water to compare the samples that contain E. coli and the BacTiter-Glo™ Reagent that was left at room temperature for different time periods.

As seen in Figure 4.61, the lysing detergent appears to still be active in the BacTiter-Glo Reagent even after being kept at room temperature for 96 hours; therefore all of the E. coli present in the sample are lysed and subsequently are inactive and not capable of reproducing. This same pattern of no growth was exhibited by samples that contained the reagent that was kept at room temperature for 72, 48, 24, and 0 hours, as well as the negative controls that contained either NB only, sterile water only, or sterile water and the reagent.

The presence of the lysing detergent may pose a problem if an attempt is made to recover and enrich bacteriophages from samples that have BacTiter-Glo

97

Reagent present because the lysing detergent will lyse the new bacterial culture in which the phages are placed.

E. coli + Sterile Water 0.300

0.250

0.200

0.150

0.100 Abs(590 Abs(590 nm)(AU) 0.050

0.000 0 5 10 15 20 25 Time (hrs)

Figure 4.60: Control Absorbance Curve for E. coli + Sterile Water

E. coli + BacTiter-Glo™ Reagent Left at Room Temp for 96 Hours 0.300

0.250 0.200 0.150 0.100

Abs(590 Abs(590 nm)(AU) 0.050 0.000 0 5 10 15 20 25 Time (hrs)

Figure 4.61: Absorbance Curve for E. coli + BacTiter-Glo Reagent Kept at Room Temperature for 96 Hours

98

4.17 E. coli and BacTiter-Glo™ Reagent Dilutions Absorbance Tests

As discussed in the previous section, the lysing detergent in the BacTiter-

Glo™ reagent does not become inactivated by being stored at room temperature for extended periods of time. Because of this, the question was raised whether or not diluting the samples that contain the reagent will allow the lysing detergent to become diluted enough so its effect does not overwhelm the entire bacterial population.

The sample containing a mixture 10-3 dilution of BacTiter-Glo and E. coli appears to be the first sample in which the bacteria growth is not completely inhibited by the lysing detergent in the BacTiter-Glo, as seen in Figure 4.62, as an increase in absorbance (growth) is observed after about 5 hours. The remaining lower dilutions and the control of E. coli and sterile water follow a relatively similar growth pattern for approximately the first 12 hours of the absorbance test, after which all the samples begin to decrease in absorbance. It is recommended that a sample that contains the BacTiter-Glo Reagent be diluted to at least 1:10,000 before it is introduced to a new bacterial culture. This may not be feasible because this also dilutes the bacteriophages that are trying to be retrieved, and also may require a very large volume of diluent.

The absorbance of the blanks over time can be seen in Figure 4.63. The absorbance for many of the blank dilutions increases rapidly after 10 hours. It is not known what caused this increase, but a hypothesis is that the light from the absorbance test interacted with the BacTiter-Glo and caused the absorbance to increase.

99

E. coli + BacTiter-Glo (Sample - Blank) Sterile Water 0.350 10^-10

10^-9 0.300 10^-8 0.250

Blank)(AU) 10^-7

- 0.200 10^-6

0.150 10^-5 10^-4 0.100 10^-3 0.050 Abs(590 Abs(590 nm)(Sample 10^-2

0.000 10^-1 0 5 10 15 20 25 Time (hrs) 10^0

Figure 4.62: E. coli and BacTiter-Glo Dilutions Growth Curves (Sample – Blank)

Blanks of BacTiter-Glo Dilutions 10^0 0.046 10^-1 0.044 10^-2 0.042 10^-3 0.040 10^-4 0.038 10^-5 0.036

10^-6 Abs(590 Abs(590 nm)(AU) 0.034 10^-7

0.032 10^-8

0.030 10^-9 0 5 10 15 20 25 30 10^-10 Time (hrs)

Figure 4.63: Absorbance of Blanks over Time

100

4.18 Retrieving Phage from a Sample Containing BacTiter-Glo™ or

Live/Dead Stain

The results presented in Figure 88 suggest that samples containing the

BacTiter-Glo reagent can be diluted and mixed with bacteria without causing a notable decrease in bacteria due to lysing via the lysing detergent. This is important in case a sample used in a bioluminescence test shows phage infection and the phages are desired to be retrieved from the sample for further use. The effects of the lysing detergent on phages were unknown, so a representative from Promega, the manufacturer of the BacTiter-Glo reagent, was contacted to obtain more information on this matter:

 It is unknown if phage can be recovered from the sample

o Lysing detergent may kill phage

o Resistance of phage to lysing detergent varies with phage

 There is no way to neutralize lysing detergent

o Technician recommended phage precipitation to isolate phage from

sample

o Technician recommended performing a titer of a phage sample

mixed with BacTiter-Glo and a sample without BacTiter-Glo

. Will lysing detergent rupture cells and give false results?

The information obtained from Promega about the BacTiter-Glo reagent raised concerns about retrieving phage from fluorescence tests that use the

101

Live/Dead Stains from Invitrogen. A representative from Invitrogen was subsequently contacted, and the following information was acquired:

 Dyes present in this stain bind to DNA of bacteria and phage

o Can cause mutation of DNA

 Dyes can detach from bacteria or phage and reattach to other cells

o Technician recommended NOT to try to retrieve phage from a

sample that has L/D stain in it to try to use it again because of

toxicity/mutagenic capabilities of the dyes

4.19 Absorbance Tests of Mixed Microbial Community with Different

Diluents

After a phage that infects S. natans is isolated, the next step in the project will be to introduce this phage to a mixed microbial community that exhibits sludge bulking caused by S. natans. A reliable method to grow the mixed community is needed, as well as the establishment of a baseline growth curve.

Each diluent shows an apparent growth pattern, as seen below in Figures

4.64a through 4.66b. Each diluent shows growth after 9 to 12 hours. This is due to the microbial community adjusting to the nutrients in the broth. In growth media, only the samples diluted in a ration of 1:100 show growth of the microbial community, but sterile water appears to have growth in all the dilutions. Of the three diluents, nutrient broth + 0.5% NaCl shows the largest change in absorbance and the most clear growth curve. Based on these results, nutrient

102 broth + 0.5% NaCl is recommended to be used for growing mixed microbial communities obtained from activated sludge.

Mixed Microbial Community + Sterile Water 0.012

0.010

Sterile Water Sterile

- 0.008 0.006 1:10^2

0.004 1:10^4 Blank (AU) 0.002 1:10^6 0.000 -0.002 0 5 10 15 20 25 30

Time (hrs) Abs(590 Abs(590 nm)of Sample

Figure 4.64a: Growth Curves for Different Concentrations of Supernatant to Sterile Water (Sample – Blank)

Mixed Microbial Community + Sterile Water 0.012

0.010

Sterile Water Sterile

- 0.008 0.006 1:10^2

0.004 1:10^4 Blank (AU) 0.002 1:10^6 0.000 -0.002 12 13 14 15 16 17 18 19 20

Time (hrs) Abs(590 Abs(590 nm)of Sample

Figure 4.64b: Expanded Window of Growth of Supernatant and Sterile Water

103

Mixed Microbial Community + Nutrient Broth + 0.5% NaCl 0.025

0.020

NB +0.5% +0.5% NB

- 0.015

0.010 1:10^2 0.005 1:10^4 Blank(AU) 0.000 1:10^6 -0.005 0 5 10 15 20 25 30 -0.010

Abs(590 Abs(590 nm)ofSample -0.015 Time (hrs)

Figure 4.65a: Growth Curves for Different Concentrations of Supernatant to Nutrient Broth + 0.5% NaCl (Sample – Blank)

Mixed Microbial Community + Nutrient Broth + 0.5% NaCl 0.025

0.020

NB +0.5% +0.5% NB

- 0.015

0.010 1:10^2 0.005 1:10^4 Blank(AU) 0.000 1:10^6 -0.005 9 11 13 15 17 -0.010

Abs(590 Abs(590 nm)ofSample -0.015 Time (hrs)

Figure 4.65b: Expanded Window of Growth of Supernatant and Nutrient Broth + 0.5% NaCl

104

Mixed Microbial Community + CYGA 0.012 0.010 0.008

0.006

CYGA CYGA Blank

- 0.004 1:10^2 0.002 1:10^4 (AU) 0.000 -0.002 0 5 10 15 20 25 30 1:10^6 -0.004 -0.006 -0.008 Abs(590 Abs(590 nm)ofSample Time (hrs)

Figure 4.66a: Growth Curves for Different Concentrations of Supernatant to CYGA (Sample – Blank)

Mixed Microbial Community + CYGA 0.012 0.010 0.008

0.006

CYGA CYGA Blank

- 0.004 1:10^2 0.002 1:10^4 (AU) 0.000 -0.002 8 9 10 11 12 13 14 15 16 1:10^6 -0.004 -0.006 -0.008 Abs(590 Abs(590 nm)ofSample Time (hrs)

Figure 4.66b: Expanded Window of Growth of Supernatant and CYGA

105

4.20 Effects of Filtering on T2 Phage Titer

Throughout this research project, samples of activated sludge were collected from various wastewater treatment plants and filtered through 0.2 µm filters in order to separate any phage that may be present from other particles in the suspension, such as bacterial cell. It was hypothesized that the filter trapped some of the phage present in the sample, even though the filter was rinsed with 1 mL of glycerin. A titer was performed on T2 phage that was filtered through a 0.2

µm filter and compared to the titer of T2 phage that was not filtered. The results for these titers are shown below in Table 4.2.

Table 4.2: Data collected from Titer of T2 Phage Plate Unfiltered T2 Plaque Count Filtered T2 Plaque Count 10^-6 A 84 61 10^-6 B 77 59 10^-6 C 74 47 Average 78.33 55.67 Std. Dev. 5.13 7.57

Average Titer 7.8 x 10^9 5.6 x 10^9

From initial inspection of the data in Table 4.2, it appears that the titer of

T2 phage is reduced by filtering. More analysis is needed in order to determine if the reduction is significant.

SPSS Software was used to perform a hypothesis test and a t-test on the above data. The results are shown below in Table 4.3.

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Table 4.3: Hypothesis Test Results

For the hypothesis test, the Null Hypothesis is ̅ = µ. The Alternative

Hypothesis is that ̅ ≠μ. As seen in Table 4.3, the p-value is 0.406 and the significance level, α, is 0.05. When the p-value is greater than α, the Null

Hypothesis cannot be rejected in favor of the Alternative. These results indicate that the reduction of the titer is not statistically significant.

Below, Table 4.4 shows the statistics that were used to perform a t-test.

Table 4.5 shows the results of this t-test. The 95% Confidence Interval (CI) for the Unfiltered T2 Plaque Count is 65.59 ≤ ̅ ≤ 91.08. The 95% CI for the Filtered

T2 Plaque Count is 36.86 ≤ ̅ ≤ 74.48. Although the CIs for both sets of data intersect, the ̅ of the Unfiltered T2 Plaque Count is not in the CI for the Filtered

T2 Plaque Count, and vice versa.

Table 4.4: One-Sample T-test Statistics One-Sample Statistics

N Mean Std. Deviation Std. Error Mean Unfiltered T2 Plaque Count 3 78.33 5.132 2.963 Filtered T2 Plaque Count 3 55.67 7.572 4.372

107

Table 4.5: One-Sample T-Test Results One-Sample Test

Test Value = 0 95% Confidence Interval of the Difference t df Sig. (2-tailed) Mean Difference Lower Upper Unfiltered T2 26.440 2 .001 78.333 65.59 91.08 Plaque Count Filtered T2 12.734 2 .006 55.667 36.86 74.48 Plaque Count

4.21 Retrieving T2 Phage from Hard Agar

Since spot tests were performed with phage filtrate on S. natans to check for the presence of phages, it was necessary to check that the titer of phage is not reduced due to phages sticking to hard agar rather than going into broth after collection of plaques.

A titer was performed on T2 phage that was mixed with hard agar and allowed to harden, homogenized with nutrient broth, centrifuged to separate the phages, then finally tittered. The results for this titer can be seen in Table 4.6 below. A titer of the T2 stock that was used in this experiment was performed in order to compare results. This titer’s results can be found in Table 4.7 below.

The titer of the stock yielded a count of 2.28x1010 pfu/mL, while the titer of

T2 phage in hard agar yielded a count of 1.77x109 pfu/mL. This is a reduction of one order of magnitude.

If phages of S. natans are isolated from a wastewater sample, they may be present in small concentrations. If this small concentration of phages is introduced to agar and retrieved in the manner in which this experiment was

108 performed, the reduction in the titer is important to consider. This emphasizes the importance of first enriching the isolated phages with S. natans before introducing agar in order to increase the phage concentration so the consequences of the titer reduction are minimal.

Table 4.6: Titer of T2 Phage in Hard Agar Plate T2 in Agar Plate Count T2 in Agar Titer 10^-6 A 159 1.59 x 10^9 pfu/mL 10^-6 B 195 1.95 x 10^9 pfu/mL 10^-6 C 178 1.78 x 10^9 pfu/mL Average 177.3 1.77 x 10^9 pfu/mL Std. Dev. 18.01 - Average Titer - 1.77 x 10^9 pfu/mL

Table 4.7: Titer of T2 Phage Stock without Hard Agar Plate T2 Stock Plate Count T2 Stock Titer 10^-7 A 236 2.36 x 10^10 pfu/mL 10^-7 B 214 2.14 x 10^10 pfu/mL 10^-7 C 234 2.34 x 10^10 pfu/mL Average 228.0 2.28 x 10^10 pfu/mL Std. Dev. 12.17 - Average Titer - 2.28 x 10^10 pfu/mL

109

CHAPTER V

CONCLUSIONS AND RECOMMENDTATIONS

As shown in the results above, infection of E. coli with both a known phage type and unknown phage types isolated from activated sludge can be detected with the absorbance, bioluminescence, and fluorescence tests by observing distinct patterns. As long as the bacteria have a good baseline growth curve, these tests repeatedly showed patterns that imply that E. coli are being lysed or inhibited due to phage infection. Further research needs to be performed to confirm whether or not these phage infection patterns can be detected with bacteria other than E. coli.

5.1 Isolation of Phages from Activated Sludge

In order to isolate phages from activated sludge samples, the following guidelines should be followed. Collect the sample and transport it to the laboratory on ice in a cooler or another container that protects the sample from light. Allow the sample to settle so there is separation of sludge and water.

Collect the desired amount of supernatant and centrifuge for 10 minutes at 1000 rpm. Collect the top liquid and filter it through a 0.2 μm filter. Note that the filtering process slightly reduces the titer of phages. Rinse the filter with 0.25 M glycine in order to collect phages that may have been trapped in the filter. Store

110 the phage filtrate samples at 4°C. When the sample is not in storage, protect it from light at all times by covering it.

After processing the activated sludge sample, the collected phages need to be enriched with S. natans in order to amplify the titer of phages that infect S. natans. A culture of S. natans should be homogenized and dispersed into aliquots along with the phage filtrate and fresh media. These samples should be kept from light in order to protect the phages and continuously shaken with oxygen exchange allowed.

5.2 Detection of Phage Infection Using the Absorbance Test

There were two patterns observed in samples that were known or presumed to have phage that imply the presence of phages that infect the bacteria being tested. These patterns should be watched for in future tests to detect phage infection in samples in which the presence of phages infected against the select bacteria is unknown. The first is an increase in absorbance the same as the control, followed by a leveling off of or decrease in absorbance for the remainder of the test period as the control’s absorbance continued to increase. The second type of pattern is a very slow increase and very low change in the absorbance compared to the control. For both patterns, the absorbance values for the phage samples at the end of the test period should be lower than the control.

The ratios that plainly showed phage infection in both the T2 and unknown phage experiments were 1000:1 and 100:1. It is recommended that the bacteria

111 concentration tested be 105 cfu/mL when using the absorbance test with samples that contain unknown titers of specific phage, because these ratios will be easier to achieve. Even if the titer is higher than presumed, phage infection will still be evidenced by the lack of absorbance increase in phage samples. Since this relatively low concentration of bacteria takes longer to grow, it is recommended that readings be taken starting 3 to 4 hours after the bacteria and phage source have been mixed together. This will allow time for the control to show growth so comparisons can be made between the control and phage samples.

5.3 Detection of Phage Infection Using the Bioluminescence Test

The bioluminescence tests yielded several clues that were interpreted as phage infection, and should be exhibited in samples that contain phages isolated from activated sludge. The first is an increase in ATP production that is higher than the control at the beginning of the test period. This increase in ATP was assumed to come from an increased demand for energy to manufacture new phages inside infected bacteria. Another sign of phage infection is a continuous decrease in the ATP after the peak has been reached, which was attributed to bacteria being inhibited or lysed due to phage infection. The peak luminescence value should be lower than the control, and should occur sooner in time. Also, the end values of ATP should be lower in phage samples than in the control.

Unlike with the absorbance test, the ratio of bacteria to phage did not seem to impact the ability to detect phage infection, nor did the concentration of bacteria. The same patterns detailed above were detected in all samples,

112 regardless of ratios or bacteria concentrations. This will be an excellent method to detect phage infection in samples in which phages are likely present in low concentrations, because the patterns can be detected even at high bacteria to phage ratios. It also eliminates the need for several different concentrations to be tested. A low concentration of bacteria (i.e. 105 cfu/mL) should be used to prepare bacteria and phage mixtures.

5.4 Detection of Phage Infection Using the Fluorescence Test

As with the other two broth tests, the fluorescence test also displays trends that are indicative of phage infection. The pattern commonly observed in samples that contain phage is an increase in the ratio of live to dead cells along with the control for the beginning of the test, then a stationary period followed by a decrease or an immediate decrease in live/dead ratio in phage samples as the controls continue to increase. In addition, the last values measured in the experiment were always lower than the control.

These trends occurred in all of the samples that contained phage in the E. coli and T2 model, indicating that the fluorescence test can show phage infection in both samples with a high bacteria to phage ratio and low bacteria to phage ratios. However in experiments with E. coli and an unknown phage from activated sludge, signs of infection were not consistent with the ratio of bacteria to phage for different concentrations of bacteria. For this reason, it is recommended that ratios of bacteria to phage of 1000:1 and 100:1 be targeted to test when searching for phages in samples isolated from activated sludge.

113

These ratios should be prepared with bacteria concentrations of 105 and 106 cfu/mL.

5.5 Next Research Steps

The next step in this project is to use the broth tests to detect phage infection of S. natans. It was shown in one experiment that growth in S. natans does not begin until about 5 hours after the absorbance test began. Because this experiment was conducted only on one occasion, it is recommended that more similar experiments be conducted to establish a baseline growth curve. In order to determine at what time point to begin readings, experiments should be performed to establish a baseline growth curve of the control at the concentration of bacteria that will be used to prepare samples of bacteria and a phage source.

The growth curves should be generated using the same test method that will be used to identify phage infection (absorbance, bioluminescence, or fluorescence).

Once a reliable growth curve is identified, the time at which growth begins to appear can be determined. After mixing bacteria with a phage source, test readings should begin at this identified time point (i.e. 5 hours after mixing bacteria and phage).

The recommended broth test for detection of S. natans phages is the bioluminescence test. Since S. natans is a slow-growing filamentous bacterium, phages in activated sludge may be present in low concentrations.

Bioluminescence tests performed on E. coli showed evidence of phage infection even at high ratios of bacteria to phage.

114

Whether using the bioluminescence or another type of test, the patterns observed in the samples that contain the phage source should be compared to the control. The control samples should exhibit the same growth trends observed in the established baseline growth curve. If the samples with the phage source exhibit this same type of growth pattern (i.e. a continuous increase), it should be concluded that phages are not present that infect the target organism. If, however, the phage samples show growth patterns that resemble those described in sections 5.2, 5.3, or 5.4 as the control continues to increase, phage infection should be concluded for these samples.

After a phage of S. natans is isolated, it should be characterized. The structure, selectivity, phage type (virulent or temperate), time to lyse the cell after absorption, and burst size of the phage are some of the important characteristics that should be determined.

Next this phage should be applied to sludge bulking that is caused by S. natans. Ratios of bacteria to phage at which the S. natans is best controlled should be determined, as well as how long after application of phage the SVI begins to reduce.

115

References

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119

APPENDIX A

SYNERGY 4™ AND GEN5™ PROTOCOLS

Absorbance Test – Used for Phage Infection Detection for Bacteria and Phage

 Medium shake for 3 seconds

 Read Absorbance at 590 nm and 630 nm

Luminescence Test using Hole/empty position on the filter wheel– Used for

Phage Infection Detection for Bacteria and Phage

 Slow shake for 3 seconds

 Incubate for 5 minutes

 Read Luminescence

Fluorescence Test using Filter-based Fluorescence Assay– Used for Phage

Infection Detection for Bacteria and Phage

 Medium shake for 1 second

 Incubate for 15 minutes

 Read Fluorescence 485 (excitation), 530(emission), 485 (excitation), 630

(emission)

Growth of S. natans Using Absorbance

 Temperature: Setpoint 30°C

 Start Kinetic [Run 24:06:00, Interval 0:30:00]

 Slow shake continuously

120

 Read Absorbance at 590nm and 630 normal read; 590 nm and 630 nm

sweep read

 End Kinetic

E. coli and BacTiter-Glo Assay Absorbance Test

 Temperature: Setpoint: 37°C

 Start Kinetic [Run 24:06:00, Interval 0:30:00]

 Slow shake for 10 seconds

 Read Absorbance at 590 nm and 630 nm

 End Kinetic

E. coli and BacTiter-Glo Assay Dilutions Absorbance Tests

 Temperature: Setpoint: 30°C

 Start Kinetic [Run 24:06:00, Interval 0:30:00]

 Slow shake for 10 seconds

 Read Absorbance at 590 nm and 630 nm

 End Kinetic

Absorbance Tests of Mixed Microbial Community with Different Diluents

 Temperature: Setpoint: 37°C

 Start Kinetic [Run 24:06:00, Interval 0:30:00]

 Slow shake for 10 seconds

 Read Absorbance at 590 nm and 630 nm

 End Kinetic

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APPENDIX B

ABSORBANCE DATA FOR E.COLI AND T2 PHAGE

Table B1: Absorbance Data for Jan 19 2010 10^3 Phage Time 0 1 2 3 4 5 10^3 Phage, 0.046 0.064 0.045 0.045 0.044 0.046 10^4 E. coli 10^3 Phage, 0.048 0.07 0.048 0.049 0.046 0.047 10^5 E. coli 10^3 Phage, 0.05 0.056 0.064 0.058 0.056 0.058 10^6 E. coli 10^2 Phage Time 0 1 2 3 4 5 10^2 Phage, 0.045 0.05 0.046 0.048 0.045 0.052 10^4 E. coli 10^2 Phage, 0.051 0.052 0.049 0.048 0.049 0.048 10^5 E. coli 10^2 Phage, 0.045 0.047 0.053 0.059 0.058 0.061 10^6 E. coli No Phage Time 0 1 2 3 4 5 No Phage, 0.045 0.046 0.046 0.056 0.044 0.045 10^4 E. coli No Phage, 0.047 0.046 0.062 0.047 0.046 0.048 10^5 E. coli No Phage, 0.049 0.05 0.063 0.054 0.055 0.076 10^6 E. coli 122

Table B2: Absorbance Data for Jan 21 2010 10^3 Phage 0 1 2 3 4 5 6 10^3 Phage, 0.045 0.042 0.044 0.046 0.045 0.047 0.046 10^5 E. coli 10^3 Phage, 0.047 0.047 0.048 0.053 0.052 0.05 0.05 10^6 E. coli 10^3 Phage, 0.053 0.058 0.068 0.081 0.081 0.084 0.063 10^7 E. coli 10^2 Phage 0 1 2 3 4 5 6 10^2 Phage, 0.043 0.045 0.046 0.046 0.046 0.047 0.046 10^5 E. coli 10^2 Phage, 0.046 0.046 0.049 0.053 0.054 0.051 0.05 10^6 E. coli 10^2 Phage, 0.051 0.058 0.069 0.087 0.088 0.091 0.07 10^7 E. coli No Phage 0 1 2 3 4 5 6 No Phage, 0.046 0.045 0.045 0.045 0.048 0.048 0.051 10^5 E. coli No Phage, 0.046 0.046 0.049 0.051 0.058 0.062 0.06 10^6 E. coli No Phage, 0.053 0.057 0.072 0.090 0.099 0.119 0.106 10^7 E. coli

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Table B3: Absorbance Data for Jan 28 2010 10^3 Phage 0 1 2 3 4 5 6 10^3 Phage, 0.065 0.063 0.088 0.145 0.161 0.418 0.498 10^5 E. coli 10^3 Phage, 0.053 0.056 0.082 0.099 0.144 0.385 0.381 10^6 E. coli 10^3 Phage, 0.064 0.093 0.110 0.122 0.125 0.168 0.226 10^7 E. coli 10^2 Phage 0 1 2 3 4 5 6 10^2 Phage, 0.050 0.056 0.068 0.085 0.152 0.217 0.281 10^5 E. coli 10^2 Phage, 0.050 0.054 0.074 0.084 0.099 0.149 0.284 10^6 E. coli 10^2 Phage, 0.062 0.088 0.118 0.14 0.129 0.154 0.178 10^7 E. coli No Phage 0 1 2 3 4 5 6 No Phage, 0.048 0.048 0.050 0.058 0.073 0.065 0.068 10^5 E. coli No Phage, 0.049 0.052 0.068 0.109 0.188 0.292 10^6 E. coli No Phage, 0.061 0.097 0.142 0.187 0.251 0.422 10^7 E. coli

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Table B4: Absorbance Data for Sept 17 2010 time 0 1 2 3 4 5 6 590 nm 10^3 Phage, 0.047 0.046 0.047 0.053 0.051 0.05 0.051 10^5 E. coli 10^3 Phage, 0.047 0.049 0.053 0.06 0.055 0.054 0.055 10^6 E. coli 10^3 Phage, 0.054 0.071 0.087 0.082 0.071 0.078 0.079 10^7 E. coli time 0 1 2 3 4 5 6 590 nm 10^2 Phage, 0.045 0.047 0.049 0.051 0.054 0.053 0.054 10^5 E. coli 10^2 Phage, 0.047 0.05 0.056 0.061 0.057 0.055 0.059 10^6 E. coli 10^2 Phage, 0.055 0.073 0.094 0.096 0.081 0.079 0.082 10^7 E. coli time 0 1 2 3 4 5 6 590 nm No Phage, 10^5 E. 0.048 0.046 0.047 0.051 0.074 0.087 0.095 coli No Phage, 10^6 E. 0.047 0.049 0.059 0.072 0.087 0.099 0.107 coli No Phage, 10^7 E. 0.057 0.074 0.095 0.101 0.109 0.118 0.131 coli

125

Table B5: Absorbance Data for Sept 22 2010 10^3 Phage Average Time 0 1 2 3 4 5 6 24 28 10^3 Phage, 10^5 E. coli 0.046 0.048 0.048 0.049 0.050 0.050 0.051 0.223 0.222 10^3 Phage, 10^6 E. coli 0.047 0.048 0.051 0.054 0.055 0.055 0.056 0.186 0.203 10^3 Phage, 10^7 E. coli 0.052 0.060 0.068 0.064 0.066 0.066 0.067 0.171 0.192 10^2 Phage Average Time 0 1 2 3 4 5 6 24 28 10^2 Phage, 10^5 E. coli 0.046 0.047 0.048 0.051 0.052 0.054 0.055 0.098 0.119 10^2 Phage, 10^6 E. coli 0.048 0.049 0.053 0.058 0.058 0.057 0.058 0.068 0.091 10^2 Phage, 10^7 E. coli 0.052 0.061 0.076 0.072 0.071 0.074 0.074 0.068 0.164 No Phage Average Time 0 1 2 3 4 5 6 24 28 No Phage, 10^5 E. coli 0.049 0.047 0.048 0.053 0.065 0.085 0.086 0.100 0.088 No Phage, 10^6 E. coli 0.047 0.049 0.053 0.074 0.087 0.090 0.103 0.301 0.312 No Phage, 10^7 E. coli 0.052 0.063 0.082 0.095 0.104 0.101 0.122 0.325 0.343

126

Table B6: Absorbance for Data Oct 4 2010 Average Absorbance time 0 1 2 3 4 5 7.5 590 nm 10^3 Phage, 0.046 0.047 0.048 0.050 0.050 0.053 0.064 10^5 E. coli 10^3 Phage, 0.047 0.048 0.053 0.057 0.057 0.061 0.070 10^6 E. coli 10^3 Phage, 0.055 0.059 0.071 0.070 0.070 0.072 0.081 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 7.5 590 nm 10^2 Phage, 0.047 0.046 0.048 0.051 0.051 0.054 0.058 10^5 E. coli 10^2 Phage, 0.048 0.048 0.056 0.057 0.057 0.057 0.057 10^6 E. coli 10^2 Phage, 0.054 0.063 0.079 0.079 0.079 0.074 0.082 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 7.5 590 nm No Phage, 0.046 0.047 0.048 0.053 0.053 0.085 0.111 10^5 E. coli No Phage, 0.048 0.048 0.057 0.074 0.074 0.094 0.121 10^6 E. coli No Phage, 0.054 0.063 0.087 0.096 0.096 0.117 0.146 10^7 E. coli

127

APPENDIX C

DATA FOR BIOLUMINESCENCE TESTS FOR E. COLI AND T2 PHAGE

Table C1: Bioluminescence Data for Jan 19 2010 10^3 Phage 0 1 2 3 4 5 10^3 Phage, 1 2 2 3 5 12 10^4 E. coli 10^3 Phage, 3 7 12 27 75 234 10^5 E. coli 10^3 Phage, 23 55 105 377 1309 2061 10^6 E. coli 10^2 Phage 0 1 2 3 4 5 10^2 Phage, 1 1 2 3 6 9 10^4 E. coli 10^2 Phage, 3 6 11 22 39 181 10^5 E. coli 10^2 Phage, 24 54 92 225 733 2632 10^6 E. coli No Phage 0 1 2 3 4 5 No Phage, 1 1 2 3 4 8 10^4 E. coli No Phage, 3 8 9 22 27 69 10^5 E. coli No Phage, 24 57 88 156 183 454 10^6 E. coli 128

Table C2: Bioluminescence Data for Jan 21 2010 10^3 Phage 0 1 2 3 4 5 6 10^3 Phage, 5 3 9 26 82 239 193 10^5 E. coli 10^3 Phage, 28 29 89 435 874 1090 784 10^6 E. coli 10^3 Phage, 289 281 587 2259 4821 2517 2002 10^7 E. coli 10^2 Phage 0 1 2 3 4 5 6 10^2 Phage, 5 4 11 23 68 239 269 10^5 E. coli 10^2 Phage, 27 32 97 224 865 2193 1139 10^6 E. coli 10^2 Phage, 284 278 605 1014 2160 3554 3881 10^7 E. coli No Phage 0 1 2 3 4 5 6 No Phage, 5 5 10 24 50 105 150 10^5 E. coli No Phage, 30 33 90 174 261 403 757 10^6 E. coli No Phage, 309 291 570 918 1186 2900 4255 10^7 E. coli

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Table C3: Bioluminescence Data for Jan 28 2010 10^3 Phage 0 1 2 3 4 5 6 10^3 Phage, 414 774 2361 4579 6528 8623 8864 10^5 E. coli 10^3 Phage, 181 411 2016 4093 4635 8586 8566 10^6 E. coli 10^3 Phage, 433 844 3197 5309 5387 10479 12305 10^7 E. coli 10^2 Phage 0 1 2 3 4 5 6 10^2 Phage, 93 272 1015 2880 4503 6508 11061 10^5 E. coli 10^2 Phage, 67 191 1244 4399 4115 5634 5177 10^6 E. coli 10^2 Phage, 317 684 1705 3262 6577 13382 22264 10^7 E. coli No Phage 0 1 2 3 4 5 6 No Phage, 4 11 54 183 902 2133 1246 10^5 E. coli No Phage, 30 84 450 884 1183 22694 10^6 E. coli No Phage, 281 596 1412 2517 21331 28657 10^7 E. coli

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Table C4: Bioluminescence Data for Sept 17 2010 Average Luminscence time 0 1 2 3 4 5 6 10^3 Phage, 10^5 E. coli 7.0 10.0 51.3 395.5 463.3 397.0 451.5 10^3 Phage, 10^6 E. coli 48.7 66.0 562.0 1007.0 1082.7 902.7 721.5 10^3 Phage, 10^7 E. coli 368.0 631.0 4856.7 5137.0 3017.0 4428.7 4678.0 Average Luminscence time 0 1 2 3 4 5 6 10^2 Phage, 10^5 E. coli 7.7 10.0 37.0 314.0 671.3 685.7 767.5 10^2 Phage, 10^6 E. coli 50.3 66.0 265.0 2200.0 1114.3 1690.3 1618.5 10^2 Phage, 10^7 E. coli 374.3 617.0 1998.0 4969.5 7445.3 12829.0 6985.0 Average Luminscence time 0 1 2 3 4 5 6 No Phage, 10^5 E. coli 7.0 11.3 51.0 229.0 956.7 2019.7 3006.5 No Phage, 10^6 E. coli 48.7 70.0 303.3 1121.0 1129.3 4751.7 9533.0 No Phage, 10^7 E. coli 469.7 675.0 1523.3 2954.5 6526.0 25308.3 24937.0

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Table C5: Bioluminescence Data for Oct 4 2010 Average Luminescence time 0 1 2 3 4 5 10^3 Phage, 10^5 E. coli 6.7 12.0 95.7 455.3 422.0 378.0 10^3 Phage, 10^6 E. coli 28.3 75.7 1013.7 817.3 913.0 1077.0 10^3 Phage, 10^7 E. coli 235.7 494.5 4595.0 3321.7 3864.0 3374.0 Average Luminescence time 0 1 2 3 4 5 10^2 Phage, 10^5 E. coli 5.0 8.7 59.0 389.7 738.7 532.0 10^2 Phage, 10^6 E. coli 27.0 56.7 632.7 1451.0 1393.7 971.0 10^2 Phage, 10^7 E. coli 258.0 597.0 3509.5 4366.0 5217.3 4505.0 Average Luminescence time 0 1 2 3 4 5 No Phage, 10^5 E. coli 4.3 9.0 61.3 216.0 856.0 1442.0 No Phage, 10^6 E. coli 24.3 66.3 346.7 960.7 1151.0 1978.0 No Phage, 10^7 E. coli 263.0 625.5 2249.0 1975.7 3694.3 10818.0

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APPENDIX D

DATA FOR FLUORESCENCE TESTS FOR E. COLI AND T2 PHAGE

Table D1: Fluorescence Data for Feb 4 2010 Ratio of 530:630 Time 0 1 2 3 4 10^3 Phage, 10^5 E. coli 1.60 1.68 2.07 2.34 2.74 10^3 Phage, 10^6 E. coli 1.89 2.80 4.06 2.37 1.57 10^3 Phage, 10^7 E. coli 4.34 9.60 10.32 7.39 6.06 Ratio of 530:630 Time 0 1 2 3 4 10^2 Phage, 10^5 E. coli 1.64 1.71 2.33 2.61 3.11 10^2 Phage, 10^6 E. coli 1.90 3.00 6.00 4.74 1.75 10^2 Phage, 10^7 E. coli 4.38 10.97 11.31 9.43 8.18 Ratio of 530:630 Time 0 1 2 3 4 No Phage, 10^5 E. coli 1.63 1.72 2.20 3.26 5.43 No Phage, 10^6 E. coli 1.88 3.13 5.61 10.28 12.49 No Phage, 10^7 E. coli 4.64 10.63 12.33 15.72 16.72

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Table D2: Fluorescence Data for Feb 22 2010 Ratio of 530:630 Time 0 1 2 3 4 5 10^3 Phage, 10^5 E. coli 1.49 1.52 1.76 2.27 2.65 2.93 10^3 Phage, 10^6 E. coli 1.65 2.00 3.65 4.02 2.01 1.51 10^3 Phage, 10^7 E. coli 3.54 6.18 11.55 8.09 7.47 5.79 Ratio of 530:630 Time 0 1 2 3 4 5 10^2 Phage, 10^5 E. coli 1.49 1.54 1.76 2.53 3.19 4.01 10^2 Phage, 10^6 E. coli 1.65 1.99 3.28 4.32 4.33 1.56 10^2 Phage, 10^7 E. coli 3.41 6.43 10.55 10.74 10.48 7.47 Ratio of 530:630 Time 0 1 2 3 4 5 No Phage, 10^5 E. coli 1.58 1.65 1.78 3.20 4.75 6.99 No Phage, 10^6 E. coli 1.77 2.08 3.39 7.90 11.26 13.11 No Phage, 10^7 E. coli 3.50 6.13 10.39 13.29 14.79 16.38

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Table D3: Fluorescence Data for Mar 2 2010 Ratio of 530:630 Time 0 1 2 3 4 10^3 Phage, 10^5 E. coli 6.41 8.74 1.65 1.84 2.03 10^3 Phage, 10^6 E. coli 2.30 2.37 2.89 3.82 4.29 10^3 Phage, 10^7 E. coli 1.70 1.62 11.68 12.56 10.51 Ratio of 530:630 Time 0 1 2 3 4 10^2 Phage, 10^5 E. coli 6.33 8.03 1.85 1.85 2.43 10^2 Phage, 10^6 E. coli 2.10 2.17 2.89 4.24 4.42 10^2 Phage, 10^7 E. coli 1.63 1.59 10.89 12.89 15.37 Ratio of 530:630 Time 0 1 2 3 4 No Phage, 10^5 E. coli 6.54 8.80 1.73 1.97 2.49 No Phage, 10^6 E. coli 2.21 2.34 3.51 5.07 9.89 No Phage, 10^7 E. coli 1.65 1.60 12.17 13.16 15.48

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Table D4: Fluorescence Data for Sept 22 2010 Average Ratio of 530:630 time 0 1 2 3 4 5 6 24 10^3 Phage, 10^5 E. coli 1.49 1.49 1.61 2.07 1.58 1.54 1.48 7.59 10^3 Phage, 10^6 E. coli 1.55 1.73 1.77 3.03 2.02 1.59 1.56 6.03 10^3 Phage, 10^7 E. coli 2.16 3.49 4.16 2.36 1.55 1.68 1.64 5.45 Average Ratio of 530:630 time 0 1 2 3 4 5 6 24 10^2 Phage, 10^5 E. coli 1.51 1.50 1.68 2.12 3.54 1.88 1.63 3.78 10^2 Phage, 10^6 E. coli 1.68 1.71 2.44 3.46 1.74 1.56 1.50 2.65 10^2 Phage, 10^7 E. coli 2.24 3.51 5.89 5.11 3.94 2.75 2.46 3.87 Average Ratio of 530:630 time 0 1 2 3 4 5 6 24 No Phage, 10^5 E. coli 1.50 1.52 1.65 2.70 5.41 8.26 8.02 4.76 No Phage, 10^6 E. coli 1.87 1.78 2.76 6.67 8.73 8.58 9.69 11.58 No Phage, 10^7 E. coli 2.26 3.77 7.67 9.54 10.20 8.85 11.46 10.73

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Table D5: Fluorescence Data for Oct 4 2010 Average Ratio of 530:630 time 0 1 2 3 4 5 7.5 10^3 Phage, 10^5 E. coli 1.59 1.60 1.80 2.29 2.97 2.03 2.29 10^3 Phage, 10^6 E. coli 1.60 1.70 2.85 3.25 2.83 2.00 2.23 10^3 Phage, 10^7 E. coli 2.45 3.54 5.44 4.72 3.60 2.22 2.18 Average Ratio of 530:630 time 0 1 2 3 4 5 7.5 10^2 Phage, 10^5 E. coli 1.55 1.60 1.78 2.43 3.01 2.36 1.75 10^2 Phage, 10^6 E. coli 1.57 1.80 3.03 2.71 2.22 1.61 1.79 10^2 Phage, 10^7 E. coli 2.40 3.83 7.22 6.82 6.81 4.95 4.28 Average Ratio of 530:630 time 0 1 2 3 4 5 7.5 No Phage, 10^5 E. coli 1.52 1.61 1.89 2.94 4.84 8.98 11.29 No Phage, 10^6 E. coli 1.65 1.91 3.65 6.94 8.29 10.03 11.71 No Phage, 10^7 E. coli 2.26 3.89 7.94 9.14 9.46 11.16 13.48

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APPENDIX E

DATA FOR E. COLI AND “TROY2 E. COLI ENRICHMENT 1”

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Table E1: Absorbance Data for Oct 24 2010 Average Absorbance time 0 1 2 3 4 5 6 590 nm 10^4 Phage, 0.045 0.045 0.049 0.055 0.044 0.046 0.045 10^5 E. coli 10^4 Phage, 0.046 0.047 0.059 0.047 0.048 0.048 0.047 10^6 E. coli 10^4 Phage, 0.055 0.067 0.080 0.059 0.057 0.050 0.053 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 6 590 nm 10^3 Phage, 0.045 0.045 0.048 0.055 0.048 0.046 0.047 10^5 E. coli 10^3 Phage, 0.046 0.046 0.067 0.050 0.049 0.047 0.049 10^6 E. coli 10^3 Phage, 0.054 0.067 0.084 0.085 0.076 0.052 0.055 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 6 590 nm 10^2 Phage, 0.044 0.044 0.049 0.055 0.053 0.046 0.089 10^5 E. coli 10^2 Phage, 0.048 0.046 0.068 0.077 0.067 0.048 0.048 10^6 E. coli 10^2 Phage, 0.055 0.065 0.086 0.100 0.095 0.057 0.066 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 6 590 nm No Phage, 0.045 0.045 0.051 0.058 0.076 0.097 0.101 10^5 E. coli No Phage, 0.046 0.047 0.070 0.087 0.087 0.098 0.109 10^6 E. coli No Phage, 0.053 0.066 0.086 0.096 0.099 0.113 0.121 10^7 E. coli

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Table E2: Absorbance Data for Nov 12 2010 Average Absorbance time 0 1 2 3 4 5 590 nm 10^4 Phage, 0.047 0.047 0.048 0.053 0.047 0.047 10^5 E. coli 10^4 Phage, 0.048 0.050 0.058 0.048 0.048 0.049 10^6 E. coli 10^4 Phage, 0.053 0.074 0.063 0.050 0.050 0.050 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 590 nm 10^3 Phage, 0.046 0.047 0.048 0.054 0.074 0.078 10^5 E. coli 10^3 Phage, 0.047 0.049 0.059 0.068 0.066 0.050 10^6 E. coli 10^3 Phage, 0.053 0.074 0.098 0.091 0.068 0.052 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 590 nm 10^2 Phage, 0.047 0.048 0.049 0.055 0.075 0.095 10^5 E. coli 10^2 Phage, 0.047 0.050 0.060 0.078 0.094 0.095 10^6 E. coli 10^2 Phage, 0.053 0.074 0.099 0.095 0.106 0.057 10^7 E. coli Average Absorbance time 0 1 2 3 4 5 590 nm No Phage, 0.049 0.050 0.049 0.058 0.079 0.106 10^5 E. coli No Phage, 0.048 0.051 0.063 0.081 0.096 0.122 10^6 E. coli No Phage, 0.054 0.076 0.102 0.099 0.120 0.146 10^7 E. coli

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Table E3: Bioluminescence Data for Oct 24 2010 Average time 0 1 2 3 4 5 6 10^4 Phage, 10^5 E. coli 9.0 20.7 315.3 1302.0 205.3 117.7 146.7 10^4 Phage, 10^6 E. coli 69.3 129.3 3625.7 611.3 362.0 260.7 267.0 10^4 Phage, 10^7 E. coli 655.3 1033.7 4453.3 2370.0 2094.0 916.7 756.7 Average time 0 1 2 3 4 5 6 10^3 Phage, 10^5 E. coli 9.0 17.0 240.0 1298.0 1516.7 418.0 336.3 10^3 Phage, 10^6 E. coli 65.3 126.7 2579.3 2141.3 1005.3 497.7 440.3 10^3 Phage, 10^7 E. coli 653.0 1027.0 2940.7 6588.7 9726.0 2328.7 2799.7 Average time 0 1 2 3 4 5 6 10^2 Phage, 10^5 E. coli 8.7 15.3 251.0 561.3 3018.0 627.3 7216.7 10^2 Phage, 10^6 E. coli 66.3 127.7 1514.0 7922.0 3186.7 648.0 689.3 10^2 Phage, 10^7 E. coli 651.7 1024.0 2997.7 3973.3 27982.3 9854.3 9482.0 Average time 0 1 2 3 4 5 6 No Phage, 10^5 E. coli 8.7 16.3 277.7 562.7 1932.3 3781.0 5707.3 No Phage, 10^6 E. coli 69.3 128.7 1335.7 1956.0 3036.0 4526.7 14726.0 No Phage, 10^7 E. coli 645.0 1026.0 2999.3 3286.3 9285.0 22378.3 25495.3

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Table E4: Fluorescence Data for Nov 12 2010 Average Ratio of 530:630 time 0 1 2 3 4 5 10^4 Phage, 10^5 E. coli 1.92 1.69 2.05 2.27 1.76 1.69 10^4 Phage, 10^6 E. coli 1.83 2.32 2.69 1.33 1.41 1.35 10^4 Phage, 10^7 E. coli 3.47 7.61 1.48 1.23 1.21 1.33 Average Ratio of 530:630 time 0 1 2 3 4 5 10^3 Phage, 10^5 E. coli 1.63 1.63 1.86 3.25 6.75 3.23 10^3 Phage, 10^6 E. coli 1.80 2.25 4.38 3.14 2.92 1.20 10^3 Phage, 10^7 E. coli 3.37 7.16 10.27 8.39 2.51 1.23 Average Ratio of 530:630 time 0 1 2 3 4 5 10^2 Phage, 10^5 E. coli 1.62 1.59 1.92 3.51 7.30 10.74 10^2 Phage, 10^6 E. coli 1.74 2.27 4.46 7.82 9.84 12.28 10^2 Phage, 10^7 E. coli 3.46 7.27 10.54 9.66 8.44 1.56 Average Ratio of 530:630 time 0 1 2 3 4 5 No Phage, 10^5 E. coli 1.58 1.61 1.99 3.64 7.93 11.06 No Phage, 10^6 E. coli 1.80 2.38 4.92 8.32 10.19 12.85 No Phage, 10^7 E. coli 3.52 7.30 10.91 10.51 12.50 14.65

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