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Sabah A.A. Jassim · Richard G. Limoges
Bacteriophages:
Practical
Applications
for Nature's
Biocontrol
Bacteriophages: Practical Applications
for Nature’s Biocontrol
Sabah A.A. Jassim Richard G. Limoges
•
Bacteriophages: Practical
Applications for Nature’s
Biocontrol
123
Sabah A.A. Jassim Richard G. Limoges
Applied Bio Research Inc. Applied Bio Research Inc.
Windsor, ON Windsor, ON
Canada Canada
ISBN 978-3-319-54050-4 ISBN 978-3-319-54051-1 (eBook)

DOI 10.1007/978-3-319-54051-1
Library of Congress Control Number: 2017932425
© Springer International Publishing AG 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt from
the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this
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authors or the editors give a warranty, express or implied, with respect to the material contained herein or
for any errors or omissions that may have been made. The publisher remains neutral with regard to
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Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer International Publishing AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
First and foremost, I give thanks to the
Almighty God for giving me the knowledge,
the strength and the direction to complete this
summary of my life’s work on
bacteriophages. I dedicate this book to my
wife Ghazal, and my three daughters:
Maryam, Sarah and Amna in thanks for their
unwavering support and understanding. In
recognition for his dedication and assistance
in articulating this work, I have named my
friend Richard Limoges as my co-author. Last
but not least, I share this information as
guidance to researchers and students who
seek a greater understanding of natural
biological processes and wish to serve
mankind with their knowledge and talents.
Sabah A.A. Jassim

Preface
We find ourselves in the twenty-first century with a world of disenchantment, a

self-imposed return to the dark ages of medicine! Most antibiotics are unable to
treat multidrug-resistant bacteria, which are causing serious diseases. Prior to the
discovery of penicillin, there were fewer bacteria that caused diseases, fewer bac-
terial mutations, less food poisoning, less water contamination. Our own inter-
ventions have caused bacterial mutations resulting in more lethal bacteria with
fewer remedies. Throughout much of the twentieth century, antibiotics have been
our primary defense against bacterial diseases. The excessive and inappropriate use
of antibiotics particularly in animal husbandry is at the root of this problem and
threatening their efficacy. The pharmaceutical industry appears unlikely to offer the
necessary countermeasures because of the objective difficulties with synthesis of
new antibiotics. The inexorable rise in the incidence of antibiotic resistance in
bacterial pathogens, coupled with the low rate of emergence of new, clinically
useful antibiotics, have encouraged researchers to revisit the bacteriophage and the
potential utility of bacteriophages in biocontrol and for preventing or treating
human and animal bacterial diseases.
The proper use of lytic ‘virulent’ bacteriophages through dietary and environ-
mental application shows promise in livestock and poultry in particular.
Bacteriophages may also be used to enhance or rekindle the effectiveness of
antibiotics in numerous applications. Bacteriophages are known to have some
advantages associated with human therapy over the use of antibiotics. However, we
urge caution since the mechanism that caused the spread of antibiotic resistance
genes between bacteria occurs most often through lysogenic
bacteriophage-mediated transduction. Inappropriate use of bacteriophages could
similarly lead to bacterial development of bacteriophage resistance. Furthermore,
bacteriophage proteins including those that are genetically modified for commercial
purposes, may also integrate into human and animal society with unknown effect.
Therefore, it would be wise to approach such methodologies with caution in order
to avoid repeating mistakes that were made with the improper use of antibiotics.
We suggest the use of properly developed and highly virulent lytic bacterio-
phages for environmental biocontrol to selectively reduce or eliminate problematic
vii
viii Preface
bacteria from sensitive environments. Bacteriophages can be effective in decon-

tamination and sanitation of both natural and manmade environments, including
farms, factories, in workplaces, crowded places, and healthcare settings or in the
laboratory. When strategically applied, they can be used without harmful effect on
and around people and animals to eliminate harmful bacteria while supporting
beneficial microflora. The ability of bacteriophages to recognize precisely their
target hosts, renders them as favorable antibacterial agents compared to
broad-spectrum antibiotics which kill target bacteria along with other beneficial
bacteria. In this book we discuss the safe use of bacteriophages as antidotes or as a
biocontrol from farm to fork and as a biodefence or to prevent biothreats while
recognizing the obstacles associated with their use.
Windsor, ON, Canada Sabah A.A. Jassim

December 2016 Richard G. Limoges
Contents
1 Enhanced Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2.1 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.2 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.3 Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.4 Preparation of FeSO4.7H2O Solution . . . . . . . . . . . . . . . . 6
1.2.5 Preparation of 13% Pomegranate Rind
Extract (PRE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2.6 Antiviral (Anti-phage) Agent (Jassim et al. 1995) . . . . . . 6
1.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.1 Bacterial Stock Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3.2 Phage Hunting/Isolation Techniques . . . . . . . . . . . . . . . . 7
1.3.3 Testing for the Presence of Crude Wild Phages (Phage
Spot Lysis Test) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.4 Production of the Transient Phage Stock
(Aldoori et al. 2015) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.5 Optimization of the Phages Lytic Characteristics
(Aldoori et al. 2015) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.6 Vertical Optimization for Phage-Host Interaction . . . . . . 9
1.4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2 Bacteriophage and Antimicrobial Resistance. . . . . . . . . . . . . . . . . . . . 19
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 The Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3 AGPs: Challenges and Consequences . . . . . . . . . . . . . . . . . . . . . . 23
2.4 AMR Shared Between Livestock and Humans . . . . . . . . . . . . . . . 25
2.5 Resistance Genes Not in a Vacuum . . . . . . . . . . . . . . . . . . . . . . . 30
2.6 Mechanisms of Gene Transfer Between Bacteria . . . . . . . . . . . . . 30
ix
x Contents
2.7 Phage, AMR and Virulence Factors in Bacteria Sharing . . . . . . . 31

2.8 Alternative to AGPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.9 Phage Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.10 Phage Therapy for Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.10.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.10.2 Disadvantages or ‘Challenges’ . . . . . . . . . . . . . . . . . . . . . 41
2.10.3 Obstacles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.11 Discussion and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3 Bacteriophage Biocontrol in Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.1.1 Poultry as a Source of Food . . . . . . . . . . . . . . . . . . . . . . 60
3.1.2 Poultry Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.1.3 Foodborne Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . 61
3.2 Sources of Campylobacter Infection for Poultry . . . . . . . . . . . . . . 62
3.2.1 Campylobacteriosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3 Sources of Salmonella Infection for Poultry . . . . . . . . . . . . . . . . . 65
3.3.1 Salmonellosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.4 Preventing Campylobacter and Salmonella in Poultry . . . . . . . . . 68
3.4.1 Campylobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
3.4.2 Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.5 Control Measures for Campylobacter and Salmonella . . . . . . . . . 71
3.6 Critical Control Points for Poultry Farms . . . . . . . . . . . . . . . . . . . 72
3.7 Ecology of Campylobacter and Salmonella Bacteriophages . . . . . 74
3.8 Phages for Biocontrol of Campylobacter and Salmonella . . . . . . . 75
3.8.1 Campylobacter Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
3.8.2 Salmonella Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.8.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.9 Approval for Direct Use on Food Products . . . . . . . . . . . . . . . . . 79
3.10 Phage-Derived Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
3.11 Challenges: Phage Biocontrol in Food Industries . . . . . . . . . . . . . 81
3.12 Phage Biocontrol from Farm-to-Fork . . . . . . . . . . . . . . . . . . . . . . 83
3.12.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
3.12.2 Phage Conceptual Applications from Farm-to-Fork . . . . . 84
3.12.3 Phage Biocontrol Applications . . . . . . . . . . . . . . . . . . . . 85
3.13 Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . 98
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4 Control, Prevention and Rapid Detection of Methicillin-Resistant
Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.2 The Occurrence of MRSA in Animals . . . . . . . . . . . . . . . . . . . . . 118
4.2.1 Household Pets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Contents xi
4.2.2 Livestock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

4.2.3 Pigs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.2.4 Poultry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.3 MRSA in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.4 MRSA Transmission Between Animals, Humans and in
Hospitals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.5 Epidemiology of MRSA in Hospitals . . . . . . . . . . . . . . . . . . . . . . 127
4.6 Decontamination of the Hospital Environment . . . . . . . . . . . . . . . 128
4.7 Bacteriophages: Nature’s MRSA Control Agents . . . . . . . . . . . . . 131
4.8 Using Phage for Decontamination of MRSA in Healthcare
Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
4.8.1 Phage Hunting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
4.8.2 Phage Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4.8.3 Phage Decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4.9 Rapid Diagnostic for MRSA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.9.1 Phage Endolysins Enzymes . . . . . . . . . . . . . . . . . . . . . . . 140
4.9.2 MRSA Urinary Tract Infections (UTIs) Detection . . . . . . 141
4.9.3 Phage Based UTI Diagnostic Kits . . . . . . . . . . . . . . . . . . 143
4.10 Conclusions and Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5 Reducing Greenhouse Gas Emissions from Livestock Farms . . . . . . . 165
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.1.1 Animal Waste the Environment, and Human Health . . . . 166
5.1.2 Natural Balance of Greenhouse Gases . . . . . . . . . . . . . . . 167
5.2 Livestock Manure Biodegradation and Production of GHGs . . . . 168
5.3 The Implication of Farm Waste in Climate Change . . . . . . . . . . . 169
5.4 Methanogenesis and Bacteriophage Interaction . . . . . . . . . . . . . . . 170
5.4.1 Methanogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
5.4.2 Biochemistry of Methanogenesis . . . . . . . . . . . . . . . . . . . 171
5.4.3 Bacteriophages (Phages) . . . . . . . . . . . . . . . . . . . . . . . . . 172
5.5 Phage Biocontrol to Reduce the Emission of GHGs in Animal
Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
5.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
6 Bacteriophage Biocontrol: Deployment in Aquatic Ecosystems . . . . . 179
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1.1 Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.1.2 Cyanobacterial Geographic Distribution . . . . . . . . . . . . . 180
6.1.3 Cyanobacterial Blooms . . . . . . . . . . . . . . . . . . . . . . . . . . 181
6.1.4 Economic and Environmental Impacts
of CyanoHABs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 181
6.1.5 Cyanophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 182
xii Contents
6.2 Cyanophage Biocontrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

6.2.1 Cyanophages Isolation and Enhancement . . . . . . . . . . . . 183
6.2.2 Phage Encapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
6.2.3 Auto-dissemination of Phages or Cyanophages . . . . . . . . 184
6.2.4 Conical Cyanophage Net (CCN) Biocontrol System . . . . 185
6.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
6.4 Conclusions ‘the Benefits’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
7 Bacteriophage Biodefense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 193
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 194
7.2 Antibiotic-Resistant Bacteria as Potential Agents of
Bioterrorism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
7.3 Bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
7.3.1 Lytic Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
7.3.2 Temperate Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
7.4 Phages as Potential Bioterrorism Agents. . . . . . . . . . . . . . . . . . . . 200
7.5 Smart Lytic Phages as Effective Anti-bioterrorism Agents . . . . . . 204
7.5.1 AMR as a Biothreat . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
7.5.2 Lysogenic Phages as Surrogates for Bacterial
Pathogens in Biothreats . . . . . . . . . . . . . . . . . . . . . . .... 210
7.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 215
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 216
8 Discussion and General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 223
8.1 Remarks and Recapitulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
8.2 The Need for Smart Lytic Phages . . . . . . . . . . . . . . . . . . . . . . . . . 224
8.3 New Wave of Super Bacteria While Antibiotics Are Failing! . . . . 225
8.4 Need to Establish New Protocols to Control Bacterial
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
8.5 Smart Phage Biocontrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
8.5.1 Human and Animal Healthcare . . . . . . . . . . . . . . . . . . . . 229
8.5.2 Animal Production and Global Warming: Mitigating
Greenhouse Gases (GHG) . . . . . . . . . . . . . . . . . . . . . . . . 232
8.5.3 Cyanobacterial Harmful Algal Blooms a Global
Problem—A Natural Solution! . . . . . . . . . . . . . . . . . . . . 233
8.5.4 Prevention of Biothreats . . . . . . . . . . . . . . . . . . . . . . . . . 235
8.6 Future Considerations for Phage Biocontrol . . . . . . . . . . . . . . . . . 237
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
About the Authors
Professor Sabah A.A. Jassim Adjunct Professor, Civil and Environmental

Engineering, University of Windsor, is CEO of Applied Bio Research Inc., Canada.
His research and academic contributions span 29 years. Sabah was awarded his
M. Phil and Ph.D. degrees from Nottingham University and Loughborough
University, respectively, both in the UK. He has worked as a faculty research fellow
and an adjunct professor at Nottingham University, UK and University of Guelph in
Canada, respectively, focussing in phage biotechnologies. He was also a visiting
professor at Universiti Putra Malaysia supervising a research postdoctoral team
working on phage design technology. Sabah has also worked extensively in the
private sector focussing on practical applications for his scientific research, espe-
cially relating to biota as well as bacteriophages and related topics.
Sabah was also once listed 13th of Power 500 the World’s Most Influential
Arabs/Middle East by Arabian Business Journal. Winner of several best research
awards, he made trend-setting achievements to the state of the art in bacteriophage
breeding and design technology to produce large-scale highly lytic phages for
biocontrol systems. These include using phages for rapid bacterial detection, rapid
drug susceptibility testing, biocontrol, alternative therapy, molecular detection and
characterization of bacterial pathogens, control of pathogens in environmental
industries, microbial bioluminescence, deletion of bacterial biofilm, bacterial stress
response, controlling harmful algal blooms and novel methods in wastewater
treatment.
His more recent innovative phage programming technology represents a model
for smart phages to gain a high-speed infection against their counterpart bacterial
pathogens which can play a significant role in decreasing bacterial pathogenic risk,
preventing loss of life and reducing the use of antibiotics in animal agriculture
industries. Dr. Jassim holds 18 international patents in several biological sciences
including phage biotechnologies. Three of these technologies have been transferred
to industrial practice. He has published extensively in prestigious journals and
conferences including peer-reviewed research and review articles as well as book
chapters and is a consistent leader in R&D to enhance bacteriophages infectious
activity to their target bacteria. Sabah has devoted much of his research to using
xiii
xiv About the Authors
phages as a novel, environmentally friendly biocontrol, particularly in agricultural

applications from farm to fork.
Mr. Richard G. Limoges currently a businessman, is operating two successful

small businesses in Windsor, Ontario where he met Sabah several years ago.
Mr. Limoges now acts as Chief Administrative Officer of Applied Bio Research,
Inc., a company dedicated to the commercialization of Dr. Jassim’s various inno-
vative technologies. Rick has a long history of community service having served for
14 years (5 terms) as a Member of Windsor City Council and Chair of numerous
Local Boards and Committees. He was next elected Member of Parliament,
Windsor-St. Clair in Canada’s 36th Parliament. Prior to his election as an M.P.,
Mr. Limoges worked as a Senior Manager in one of Canada’s largest banks. Rick is
a graduate of the University of Windsor with Honours in Business Administration,
and has applied his communication skills to assist Dr. Jassim in disseminating his
research and life’s work into several peer-reviewed publications in scientific jour-
nals and now this book. In recognition of Rick’s efforts and dedication to assisting
Dr. Jassim with this work, he is honored to be named as co-author in several of
Sabah’s publications.
Acronyms and Abbreviations
AAP American Academy of Pediatrics

ABHRs Alcohol-Based Hand Rubs
AGPs Antibiotic Growth-Promoters
AMR Antimicrobial Resistance
ATP Adenosine Triphosphate
BGA Blue-Green Algae
BoNT Clostridium botulinum Neurotoxin
BSL-2 Biosafety Level-2
BTA Biothreat Alarm System
BZ Burst Size
CA Community-Associated
CCN Conical Cyanophage Net
CDC Centers for Diseases Control and Prevention
CFR Code of Federal Regulations
CFU Colony-Forming Units
CHAPK Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase
CoNS Coagulase-Negative Staphylococci
CoPS Coagulase-Positive Staphylococci
CRE Carbapenem-Resistant Enterobacteriaceae
CyanoHABs Cyanobacterial Harmful Algal Blooms
DFPS Dry Fog Phage System
DNA Deoxyribose Nucleic Acid
ECDC European Centre for Disease Prevention and Control
EFSA European Food Safety Authority
EHEC Enterohemorrhagic E. coli
ELISA Enzyme-Linked Immunosorbent Assay
EMRSA Epidemic MRSA
EPA Environmental Protection Agency
ESBL Multidrug-Resistant Extended-Spectrum b-Lactamase
ESR Institute for Environmental Science and Research
xv
xvi Acronyms and Abbreviations
EU European Union
FAO Food and Agriculture Organization
FDA Food and Drug Administration
FIGE Field Inversion Gel Electrophoresis
FnBPA Fibronectin-Binding Protein A
FSEP Food Safety Enhancement Program
FSIS Food Safety Inspection Services
GFP Green Fluorescent Protein
GHG Greenhouse Gas
GMP Good Manufacturing Practices
GRAS Generally Recognized as Safe
HA Hospital-Associated or Acquired
HABs Harmful Algal Blooms
HACCP Hazard Analysis Critical Control Point
HCW Healthcare Worker
HGT Horizontal Gene Transfer
HICPAC Healthcare Infection Control Practices Advisory Committee
ICMSF International Commission for Microbiological Safety of Foods
IDSA Infectious Diseases Society of America
IR Infective Ratio
LA Luria Agar
LA-MRSA Livestock-Associated MRSA
LB Luria Broth
LEAD Livestock, Environment and Development
LODs Limits of detections
LuxAB-PASA
LuxAB-Phage Anthracis Spore Alarm
MDRB Multidrug-Resistant Bacteria
MRSA Methicillin-Resistant Staphylococcus aureus
MRSP Methicillin-Resistant Staphylococcus pseudintermedius
MSSA Methicillin-Susceptible Staphylococcus aureus
NDM New Delhi Metallo-b-Lactamase
OIE World Organisation for Animal Health
PAD Phage Alarm and Detector
PBHR Phage-Based Hand Rubs
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PDA Phage Alarm and Detector
PFGE Pulsed-Field Gel Electrophoresis
PFU Plaque Forming Units
PIA Polysaccharide Intercellular Adhesion
PLPs Phage-Like Particles
PRE Pomegranate Rind Extract
PVL Panton-Valentine Leukocidin
Q&Q Qualitative and Quantitative
Acronyms and Abbreviations xvii
QRA Quantitative Risk Assessment

RNA Ribonucleic Acid
SE Salmonella enterica serovar Enteritidis
SEs Staphylococcal Enterotoxins
SHEA Society for Healthcare Epidemiology of America
STEC Shiga-Toxin producing E. coli
SUR Solar Ultraviolet Radiations
UK United Kingdom
US FDA United States Food and Drug Administration
USA United States of America
USDA United States Department of Agriculture
USDA-FSIS United States Department of Agriculture- Food Safety
and Inspection Service
US-FSIS US-Food Safety and Inspection Service
UTIs Urinary Tract Infections
UV Ultraviolet
VFA Volatile Fatty Acids
WHO World Health Organization
Chapter 1
Enhanced Bacteriophages
Abstract The bacterial cell wall is the most important part of the bacterial structure
for bacteriophage attachment, which is required to initiate infection. The rapid and
precise attachment of the phage onto a susceptible host cell is the first step of
infection. In this chapter, methods are described to control phage-host interactions
and to produce highly lytic phages with no or far less phage-resistant mutants, along
with broad host targeting capabilities. These methods do not employ genetic
modification to breed ‘re-tailored’ wild phages with auxiliary mechanisms for
phage adherence, adsorption, binding and uptake which are critical for plaque
formation. The purpose of these tactics is to gain new sub-strains of phages that are
able to infect previously resistant bacteria and to play an important role in future
applications.
Keywords Bacteriophage Phage design Phage breeding Phage reprogram-

ming technology
Contents
1.1 Introduction........................................................................................................................ 2
1.2 Materials ............................................................................................................................ 4
1.2.1 Media ...................................................................................................................... 5
1.2.2 Buffers..................................................................................................................... 5
1.2.3 Bacteria ................................................................................................................... 6
1.2.4 Preparation of FeSO4.7H2O Solution..................................................................... 6
1.2.5 Preparation of 13% Pomegranate Rind Extract (PRE).......................................... 6
1.2.6 Antiviral (Anti-phage) Agent (Jassim et al. 1995) ................................................ 6
1.3 Methods ............................................................................................................................. 7
1.3.1 Bacterial Stock Culture .......................................................................................... 7
1.3.2 Phage Hunting/Isolation Techniques...................................................................... 7
1.3.3 Testing for the Presence of Crude Wild Phages (Phage Spot Lysis Test) ........... 8
1.3.4 Production of the Transient Phage Stock (Aldoori et al. 2015) ........................... 8
1.3.5 Optimization of the Phages Lytic Characteristics (Aldoori et al. 2015)............... 9
1.3.6 Vertical Optimization for Phage-Host Interaction ................................................. 9
1.4 Notes .................................................................................................................................. 16
References .................................................................................................................................. 18
© Springer International Publishing AG 2017 1

S.A.A. Jassim and R.G. Limoges, Bacteriophages: Practical Applications
for Nature’s Biocontrol, DOI 10.1007/978-3-319-54051-1_1
2 1 Enhanced Bacteriophages
1.1 Introduction
Bacteriophage (phage) interactions with bacteria can be divided into two broad life
cycles based on the biological outcome of infection. The lysogenic cycle, also
called ‘temperate’ or ‘dormant’ typically integrates phage DNA within the host
bacteria. The prophage “endogenous phage”, or latent form of phage, also called
lysogens (this is discussed in greater detail in Chap. 2), has a circular form of the
phage’s genome, crossing over between the prophage genome and the circular
bacterial genome (Jassim and Limoges 2014). This allows prophage genome to
replicate along with the bacterial genome, also protecting the bacterium from fur-
ther infection. The phage’s genome in this state is called a prophage and during this
time, the bacteria may appear to be dividing in phage-free conditions. The phage
and bacterial genomes will remain integrated until the prophage is induced or
activated by adverse conditions to then proceed to cause lysis (Jassim and Limoges
2014). Typical plaques made by lysogenic phage lambda are pinpoint turbid pla-
ques, due to lysogenization of bacteria within the plaque.
The phage lytic cycle involves a host-specific parasitic relationship in which the
phage uses the energy and the metabolic machinery of a bacterium for reproduction.
Unlike the lysogenic cycle, they do not integrate their genetic material into the host
cell chromosome and usually the host cell undergoes lysis and dies, simultaneously
liberating a large number of progeny phages, which are each then ready to start
another cycle by infecting the surrounding bacteria (Jassim and Limoges 2014).
The lytic phage fits in the class of ‘natural living antimicrobial agents’ and are
arguably the most abundant biological entities on the planet (Jassim and Limoges
2014). Most lytic cycles take between 30 and 40 min depending on factors such as
bacterial growth conditions, target bacterial type, phage host-interactions, phage
infective ratio and phage biokinetic values, such as phage binding or adsorption
time, phage amplification, burst time and burst size (Jassim and Limoges 2013,
2014). Understanding these phage-host interactions is very important for the pro-
gramming of successful bacterial pathogen biological controls. If lysis occurs too
quickly, it will not produce enough new phages to infect neighbouring bacterial
cells. If it is too slow, the phage loses an opportunity to infect new host cells for
further replication, perhaps giving the bacterial cell time to adapt and become
resistant to the phages. This bacterial adaptation, aided by the influence of envi-
ronmental forces (Jassim and Limoges 2013), may also give rise to the lysogenic
cycle, allowing phage DNA to integrate into the host DNA (see above). If extra-
cellular phage concentrations are too high in this regard some lytic phages undergo
a phenomenon known as lysis inhibition, where completed phage progeny will not
immediately lyse out of the cell. This mechanism is not identical to that of tem-
perate phage going dormant and is usually temporary.
Phages and bacteria are continually and rapidly evolving in the ecosystem, with
bacteria becoming phage-resistant and phages evolving to maintain or improve
infectivity of host bacteria (Jassim and Limoges 2014; Maxwell 2016). It may
therefore be better to view phages as not merely parasitic on the host cells but as
1.1 Introduction 3
opportunistic biological, living agents where survival depends not only on the host
cell for its propagation but is also influenced by several environmental biophysical
factors including virophages, nutrients (macronutrients and micronutrients), tem-
perature, light, solar UV radiation, pH, cation (+ve ion) concentrations, salinity,
seasonal variations, microbial community and other environmental forces (Jassim
and Limoges 2013, 2014). These factors can either impede or expedite phage–host
interactions and the resultant viral infection and killing of the bacterial cell, which is
a necessary outcome of successful phage therapies and biocontrols using lytic
phages (Jassim et al. 2012; Abdulamir et al. 2014; Jassim and Limoges 2014).
Phages that are best equipped to survive and reproduce perpetuate the highest
frequency of their dominant genes to descendant populations. This is the principle
known colloquially as ‘survival of the fittest,’ where fitness denotes the overall
ability to pass copies of phage genes on to successive generations within their
bacterial communities. The evolutionary survival of phages is attributed to five
factors: genetic variability, variety in means of transmission, efficient replication
within host cells, ability to remain dormant within the host (lysogeny), and envi-
ronmental or external forces (Jassim and Limoges 2014).
Based on the above concepts, phage vertical breeding protocol was developed by
Jassim et al. (1995) to select and breed the best phage to target host cells. This is a
method of selecting a virulent phage from a population and breeding the selected
phage to create more assertive progeny. Steps include:
(i) selecting a target cell sample of the test phage;
(ii) incubating a mixture of this phage population with the target cell sample so
as to effect attachment to and infection of target cells by test phage particles;
(iii) incubating the target cells to complete infection by the test phage and to
cause phage progeny to be released, then recovering the phage progeny;
(iv) an additional step performed between steps (ii) and (iii) comprises neutral-
izing the extracellular phage with an antiviral agent in order to control the
conditions of the exercise and to select only the most virulent phages.
Horizontal phage design technology (Jassim et al. 2010) was developed to
address phage-host interactions and to produce highly lytic phages with no or far
less phage-resistant mutants, along with broad host targeting capabilities. Both
methods, phage vertical breeding (Jassim et al. 1995) and horizontal phage design
(Jassim et al. 2010), do not employ genetic modification to breed “re-tailored” wild
phages on the host cells in order to gain newly bred sub-strains of phages which are
able to overcome the host defence mechanisms in order to infect previously
resistant bacteria and to play an important role in future applications (Jassim et al.
1995, 2012; Hibma et al. 1997; Abdulamir et al. 2014; Jassim and Limoges 2014).
Recently we have developed newer methodologies to reprogram phages to
possess (Fig. 1.1) (Jassim and Limoges 2014) auxiliary mechanisms for phage
adherence/adsorption/binding and uptake that are critical for plaque formation, in
order to gain new sub-strains of phages able to infect previously resistant host cells,
namely prophage. This non-genetic approach of the technology is
Fig. 1.1 Non-genetic phage programming technology to produce smart lytic phage. Source
Applied Bio Research Inc.; Jassim and Limoges (2014)
environmentally-driven and so mimics natural selection or evolution of the phage

by reproducing vast numbers of mixed populations of the most robust wild-type
phages. Phage programming technology, illustrated in Fig. 1.1, remains a propri-
etary technology which permits a better selection and adaptation of robust lytic
phages for each potential application. This technology is capable of converting
naturally occurring wild phages including prophages to smart phages with a broader
range of host specificity that can overcome a bacterium’s resistive defense mech-
anisms. Finally, several suggestions are shared in order to give direction for
overcoming common obstacles in applied phage technology.
The key to successful use of phages in modern scientific, farm, food processing
and clinical applications is to understand the common obstacles as well as best
practices and to develop answers that work in harmony with nature. Here, we
describe various methodologies for adapting phages with enhanced virulent prop-
erties; such approaches have been extensively modified from our earlier published
work with vertically bred phages (Jassim et al. 1995).
1.2 Materials
All media, solutions and buffers are prepared using water distilled from drinking
water and analytical grade reagents. Prepare and store all media, solutions and
buffers at room temperature (unless indicated otherwise). Diligently follow all
1.2 Materials 5
waste disposal regulations when disposing waste materials. Work with moderately
hazardous bacteria should be done in a biosafety level-2 (BSL-2) facility.
A laboratory coat or gown and gloves should be worn. There should be no mouth
pipetting, eating, or drinking in the laboratory and materials used in laboratory
(pipettes, slides, culture vessels, etc.) as well as work surfaces should be decon-
taminated. In working with hospital and other environmental samples, it should be
remembered that these can carry other dangerous pathogenic organisms and
therefore, must be conducted in a biological safety cabinet for level 2 organisms.
Laboratory infections can occur as a result of splashes and failure to wash hands
after handling potentially infectious materials. It is recommended that laboratory
personnel be offered the vaccine for cretin bacterial pathogens. Vaccine will not
decrease the risk of infection but can reduce the potential health risk of
laboratory-acquired disease.
1.2.1 Media
1. Luria broth (LB): tryptone 10 g l−1 (HiMedia, Mumbai, India), yeast extract
5 g l−1 (HiMedia, Mumbai, India), and sodium chloride 10 g l−1 (HiMedia,
Mumbai, India) adjust at pH 7.2.
2. Luria-agar (LA) consisted of the above with the addition of 14 g l−1 agar
(HiMedia, Mumbai, India) use for culture maintenance.
3. Bacterial dilutions from 18 h LB cultures grown at 37 °C to be carried out in
phosphate buffered saline (PBS, Oxoid, UK).
4. Top layer agar: Prepare LB with Lambda-buffer supplemented with 4 g l−1 agar
bacteriology No. 1 (No. 1 Oxoid).
1.2.2 Buffers
1. PBS: Dissolve one tablet in 100 ml of a 1X solution. 1X PBS solution contains

10 mM phosphate buffer, 137 mM sodium chloride, and 2.7 mM potassium
chloride. Each tablet prepares a 1X PBS solution when dissolved in 100 ml of
distilled water H2O. Adjust the pH to 7.2 with HCl. Dispense the solution into
aliquots and sterilize them by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on
liquid cycle or by sterile filter membrane 0.45 lm units. Store PBS at room
temperature.
2. Lambda-buffer: Prepare from 6 mmol l−1 Tris pH 7.2, 10 mmol l−1 Mg
(SO4)2.7H2O, 50 lg ml−1 gelatin (Oxoid, UK). Adjust the pH to 7.2 with HCl.
Dispense the buffer into aliquots and sterilize them by autoclaving for 20 min at
15 psi (1.05 kg/cm2) on liquid cycle or by Sterile filter membrane 0.45 lm
units. Store PBS at room temperature.
1.2.3 Bacteria
Phage cultures require host cells (bacteria) in which the phages multiply. The
standard reference bacterial culture can be obtained from microbial culture col-
lections. The target bacteria can also be isolated from animal farms, hospital, soil,
mud, sewage disposals, etc.
1.2.4 Preparation of FeSO4.7H2O Solution
1. First freshly prepare solution (0.01%) of FeSO4.7H2O in Lambda-buffer, pH 6.3.

2. Sterilized by membrane filtration (0.45 lm, Whatman).
1.2.5 Preparation of 13% Pomegranate Rind Extract (PRE)
1. Blend pomegranate rind in distilled water (25% w/v) and boil for 10 min.
2. Centrifuge (20,000g, 4 °C, 30 min) (see Note 1.4.1).
3. Autoclave supernatant (121 °C, 15 min) and cool.
4. Store at −20 °C.
5. Prior to use, mix 1.3 ml of stock solution of PRE (25% w/v) with 8.7 ml of
Lambda buffer. The final PRE concentration is 13% (see Note 1.4.2).
1.2.6 Antiviral (Anti-phage) Agent (Jassim et al. 1995)
Immediately prior to use, transfer 3.3 ml of 13% PRE added to 7 ml of freshly

prepared ferrous sulphate solution (0.01%). After about 30 s the colour of the
mixture changes to greenish then to black. These mixtures of ferrous sulphate and
PRE should be protected from light. The mixture is active for 45 min after
preparation.
1.3 Methods 7
1.3 Methods
Carry out all procedures at room temperature unless otherwise stated.
1.3.1 Bacterial Stock Culture
1. Prior to procedure, a stock culture of the phage target bacteria should be

maintained on LA plate (see Note 1.4.3).
2. Inoculate 1 loopful of the bacteria into a 100 ml Erlenmeyer flask containing 10 ml
of LB (supplemented with 10 mmol l−1 CaCl2.2H2O) at pH 7.2 (see Note 1.4.4).
3. Incubate for 18 h at 37 °C in an orbital shaker [200 revolutions per minute (rev
min−1)].
4. One ml serial dilutions prepare in LB (supplemented with 10 mmol l−1
CaCl2.2H2O).
5. The cell concentrations used in this study are estimated as 101, 102, 103, 104,
105, 106 and 107 colony-forming units per millilitre (cfu ml−1).
1.3.2 Phage Hunting/Isolation Techniques
1. Collect crude specimens of approximately 50 g of environmental dirt, sewage

disposal, and poultry or cattle waste in a sterile sample collection tube (100 ml).
2. Mix each specimen thoroughly and transfer 5 g of each specimen into 90 ml of
LB in 250 ml Erlenmeyer flask with cotton-plug and mix using a vortex mixer
for 30 s.
3. Then add 5 ml of 10 h LB cultures of the target bacterial isolate or reference
strains and mix by a vortex mixer for 30 s then incubate at 37 °C in an orbital
shaker (50 rev min−1).
4. After 18 h, withdraw 20 ml of the mixture into a sterile 50 ml test tube, cell
debris is pelleted by centrifugation at 5000g at room temperature for 5 min.
5. Divide the supernatant into two portions (2 10 ml). Transfer each 10 ml
portion into 2 sterile 15 ml test tubes:
(i) Add to one portion of 10 ml supernatant, 1 ml of chloroform (Sigma,
USA) with gentle shaking of tubes for 5 min then incubate on crushed ice
for 5 min. A milky solution appears due to bacterial protein digestion by
chloroform. Bacterial cell debris and bacterial ghosts are discarded by
centrifugation at 5000g for 5 min at room temperature. Collect the top
aqueous supernatant into 15 ml sterile tube and store at 4 °C as a possible
phage solution and add 5 ll of chloroform to prevent microbial
contamination.
(ii) Filter the second portion of 10 ml supernatant via sterile membrane filters
25 mm diameter 0.45 nm pore size (Millipore) then by 25 mm diameter
0.25 nm pore size (Millipore). Collect the filtrate solution into a 15 ml
sterile tube and store at 4 °C as a possible phage solution and add 5 ll of
chloroform to prevent microbial contamination.
1.3.3 Testing for the Presence of Crude Wild Phages

(Phage Spot Lysis Test)
1. Prepare bacterial lawns of target bacterial isolates or reference bacterial strains

by adding 500 µl of LB 18 h cultures on LA plates, allowing the liquid bacterial
culture to soak into the LA, with a half lid cover on the plate, at room tem-
perature or incubator at 37 °C for 20 min (see Note 1.4.5).
2. Transfer 10 µl of the possible phage solution on the bacterial lawns and then
incubate at 37 °C.
3. Check for plaques or lysis spots which are observed after 6–18 h. The detection
of phage presence is based on visual appearance of a lysis zone at the site where
the 10 µl solution was added onto the surface of the target bacterial lawn.
Positive results are expressed by either clear or semi-clear (turbid) lysis zones,
while negative results are expressed by the absence of such lysis zones.
1.3.4 Production of the Transient Phage Stock

(Aldoori et al. 2015)
1. Cut 5–10 plaques with a sterile Pasteur pipette and place them in 1.5 ml sterile
Eppendorf micro-centrifuge tubes (polypropylene; 1.5 ml; Sarstedt).
2. Add into 300 µl of Lambda buffer and incubate for 30 min at room temperature
with intermittent gentle shaking every 10 min.
3. Add 1:10 chloroform:lysate ratio with gentle shaking for 10 min at room tem-
perature in order to elute the phages from the agar to lyse the bacterial cells.
4. Incubate the mixture for 3 min in crushed ice.
5. Cell debris is removed by centrifugation at 5000g for 15 min at room tem-
perature and the supernatant is transferred to a 1.5 ml sterile Eppendorf
micro-centrifuge tube.
1.3 Methods 9
1.3.5 Optimization of the Phages Lytic Characteristics

(Aldoori et al. 2015)
1.3.5.1 Plaque-Based Optimization
The isolates of wild lytic phages from the transient stocks are propagated with the
corresponding host bacterial isolates and the representative standard reference
bacterial strains using the plate method as follows:
1. Prepare 10-folds serial dilutions (10−1–10−7) with Lambda buffer for the phage
stock solutions by taking 100 µl of the phage solution into 900 µl of lambda
buffer.
2. Transfer of 100 µl of each dilution for each phage stock solution into 15 ml
volume sterile plastic containers containing 100 µl of 109 cfu ml−1 of 18 h
bacterial host LB culture and incubate at 37 °C.
3. After 10 min incubation add 2.5 ml of top layer agar cooled to 45 °C and
poured over LA plates and incubate at 37 °C for 18–24 h.
4. Plaques morphology and growth characteristics are recorded according to the
following parameters:
(i) plaque visible time,
(ii) clarity or turbidity of the plaque,
(iii) diameter (mm) of the plaque,
(iv) shape of the plaque,
(v) depth of the plaque,
(vi) margin cut.
1.3.5.2 Phage Enhancement of Lytic Activity
1. Select the best plaques according to the above categories in Sect. 1.3.5.
2. Repeat step in Sect. 1.3.5.1 several times in order to magnify the outcome of the
biased selection of the larger and clearer plaques each time, until obtaining the
largest and clearest plaques, which represent the upper limits of plaque-based
optimization, reflecting the best possible enhancement of the lytic characteristics
of the phages.
1.3.6 Vertical Optimization for Phage-Host Interaction
Use the phage breeding protocol, modified from our earlier published data (Jassim
et al. 1995) to increase the vertical biokinetic interaction of the phages resulting
from the steps in Sect. 1.3.5.2, for their host cell. In brief, this process will increase
Fig. 1.2 Principle of vertical optimization for phage-host interaction
the virulence of the isolated phages within their bordering host population
(Fig. 1.2).
This process uses an antiviral compound derived from pomegranate extract
mixed with ferrous sulphate, which destroys phages that are free in suspension but
not those that are bound to a host cell surface. When added to broth containing both
phage and host bacteria, the process will select those phages that adsorb most
rapidly, as the unbound or slower phages will be destroyed. The progeny from the
above infections will be passed through repeated rounds of this breeding process to
produce the ultimate surviving progeny phage that has demonstrated enhanced
virulence.
1.3.6.1 Phage Mass-Action Biokinetics to Select the Elite Phages
1. Place 200 ll of an appropriate Lambda dilution of bacterial host cell 105 cfu ml−1
in a sterile Eppendorf micro-centrifuge tube.
2. Add phage (see Sect. 1.3.5.2) 200 ll of the highest phage titer (109–1012 pfu ml−1)
(where pfu represents plaque forming units per ml) in Lambda buffer. Mix gently to
avoid causing any bubbles.
1.3 Methods 11
3. At appropriate contact time 2, 5, 7, 10, and 15 min transfer 20 ll from

phage-bacteria mixture into a sterile Eppendorf micro-centrifuge tube.
4. Add 150 ll of the PRE/FeSO4 solution (Antiviral Agent see Sect. 1.2.6) and
mix gently to avoid causing any bubbles.
5. Incubate for 5 min at room temperature.
6. The activity of the virucidal agent is neutralized by adding an equal volume
(150 ll) of 2% (v/v) Tween-80 (Sigma Chemical Co., St Louis, MO, USA) in
Lambda buffer.
7. After 30 s transfer the mixture into 15 ml sterile tube and add 680 µl of 109
cfu ml−1 of 18 h bacterial host LB culture and mix.
8. Immediately add 2.5 ml of top layer agar cooled to 45 °C and pour over LA
plates and incubate at 37 °C for 18–24 h.
9. Plaques morphology and growth characteristics are recorded according to the
following parameters:
(i) plaque visible time,
(ii) clarity of the plaque,
(iii) diameter (mm) of the plaque,
(iv) shape of the plaque,
(v) depth of the plaque,
(vi) margin cut.
1.3.6.2 Propagation of the Elite Phages
1. Select the best plaques according to the morphology and growth characteristics
above in Sect. 3.6.1.
2. Cut the plaques using a sterile Pasteur pipette and place them in 1.5 ml sterile
Eppendorf micro-centrifuge tube. Each plaque will contain approximately 107
pfu ml1.
3. Add into 300 µl of Lambda buffer and incubate for 30 min at room temperature
with intermittent gentle shaking every 10 min.
4. Add 1:10 chloroform:lysate ratio with gentle shaking for 10 min at room tem-
perature in order to elute the phages from the agar and to lyse the bacterial cells.
5. Incubate the mixture for 3 min in crushed ice.
6. Centrifuge at 5000g for 15 min at room temperature and transfer the super-
natant into a 1.5 ml sterile Eppendorf micro-centrifuge tube and store at 4 °C as
a phage stock solution.
1.3.6.3 Phage Titer
1. Make 10-fold serial dilutions of the selected phage in lambda buffer.

10−1 dilution is 10 ll of selected phage particles from the phage stock solution
into 990 ll of lambda buffer; mix by pipetting up and down,
10−2 dilution is 10 ll of 10−1 dilution into 990 ll of lambda buffer; mix as
above.
10−3 dilution is 10 ll of 10−2 dilution into 990 ll lambda buffer; mix as above.
2. Transfer 100 µl of each dilution for each phage stock solution into a 15 ml
volume sterile plastic container contain 100 µl of 109 cfu ml−1 of 18 h bacterial
host LB culture and incubate for 15 min at 37 °C.
3. Add 2.5 ml of melted supplemented (10 mmol l−1 MgSO4) LB top agar (cooled
to *48 °C). Vortex gently and pour onto pre-warmed (37 °C) LA plates. Allow
the top agar to solidify and then incubate overnight at 37 °C for 18 h.
4. Count the plaques and determine the titer (pfu ml−1) and packaging efficiency
(See sample calculations).
1.3.6.4 Phage Titer Calculations
If there were 35 plaques on a 10−7 dilution plate, then the phage titer, pfu ml−1, of
this reaction would be:
ð# of plaquesÞ ðdilution factorÞ ð1000 ll=mlÞ

ðvolume of phage plated ðllÞÞ
ð35 pfuÞ ð107 Þ ð1000 ll=mlÞ

For this example : ¼ 3:5 109 pfu ml1
ð100Þ
1.3.6.5 Improve Phage Adsorption ‘Attachment’ Time
To optimize the time for the encounter between bacterial hosts and their specific
phages giving the highest number of phage particles at the pre-burst era or yielding
the highest infective ratio.
1. Place 200 ll of an appropriate Lambda dilution of bacterial host cell 105 cfu ml−1
in a sterile Eppendorf micro-centrifuge tube.
2. Add 200 ll of the selected elite (see Sect. 1.3.6.2) from the highest phage titer
in Lambda buffer. Mix gently to avoid causing any bubbles.
3. At phage-host contact time intervals 1, 2, 3, 4, and 5 min transfer 20 ll from
phage-bacteria mixture into a sterile Eppendorf micro-centrifuge tube.
1.3 Methods 13
4. Add 150 ll of the PRE/FeSO4 solution (Antiviral Agent see Sect. 1.2.6) and mix
gently to avoid causing any bubbles then incubate at room temperature for 5 min.
Lambda buffer.
6. After 30 s transfer the mixture into 15 ml sterile tube and add 680 µl of 109 cfu ml−1
of 18 h bacterial host LB culture and mix.
plates then incubate at 37 °C for 18 h.
8. Plaques morphology, growth characteristics are recorded as above.
9. Repeat Sects. 1.3.6.2, 1.3.6.3 and 1.3.6.4.
1.3.6.6 Infective Ratio (IR)
The ratio between the number of phage particles at the pre-burst era and the number
of the bacterial hosts used in the assay is the infective ratio (IR).
1. Add 200 ll of an appropriate Lambda dilution of bacterial host cells 105 cfu ml−1
in sterile Eppendorf micro-centrifuge tubes.
2. Add 200 ll of the selected elite phage (according to Sects. 1.3.6.2 and 1.3.6.5)
at titer 105, 106, or 107 pfu ml−1 in Lambda buffer to each tube containing the
host cells. Mix gently to avoid causing any bubbles.
3. After the contact time (see Sect. 1.3.6.5), for example 3 min, transfer 20 ll from
each phage-bacteria mixture into a sterile Eppendorf micro-centrifuge tube.
4. Add 150 ll of the PRE/FeSO4 solution (Antiviral Agent see Sect. 1.2.6) and
mix gently to avoid causing any bubbles then incubate at room temperature for
5 min.
Lambda buffer.
6. After 30 s transfer the mixture into 15 ml sterile tube and add 680 µl of 109
cfu ml−1 of 18 h bacterial host LB culture and mix.
plates and incubate at 37 °C for 18 h.
8. Phage titer calculations (Sect. 1.3.6.4).
9. Calculate the IR
# of phage particles in the pre burst era at a given dilution

¼
# of the bacterial hosts used in the assay at the same given dilution
6 pfu 106 ml1
or : ¼ 60
cfu 105 ml1
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Commencement in 1769, in fabrics of American homespun
manufacture. The senior class of the previous year at Harvard had
been similarly dressed.
These little bands of patriotic women gathered far and wide
throughout New England. At one meeting seventy linen wheels were
employed. In Newbury, Beverly, Rowley, Ipswich, spinning matches
were held. Let me show how the day was spent. I quote from the
Boston News-Letter:—
Rowley. A number of thirty-three respectable ladies of the
town met at sunrise [this was in July] with their wheels to
spend the day at the house of the Rev’d Jedidiah Jewell in the
laudable design of a spinning match. At an hour before
sunset, the ladies then appearing neatly dressed, principally
in homespun, a polite and generous repast of American
production was set for their entertainment, after which being
present many spectators of both sexes, Mr. Jewell delivered a
profitable discourse from Romans xii. 2: Not slothful in
business, fervent in spirit, serving the Lord.
You will never find matters of church and patriotism very far apart
in New England; so I learn that when they met in Ipswich the
Daughters of Liberty were also entertained with a sermon. The
Newbury patriots drank Liberty Tea, and listened to a sermon on the
text Proverbs xxxi. 19. Another text used at one of these gatherings
was from Exodus xxxv. 25: “And all the women that were wise-
hearted did spin with their hands.”
The women of Virginia were early in the patriotic impulses, yet few
proofs of their action or determination remain. In a Northern paper,
the Boston Evening Post of January 31, 1770, we read this Toast to
the Southerners:—
NEW TOASTS.
The patriotic ladies of Virginia, who have nobly
distinguished themselves by appearing in the Manufactures of
America, and may those of the Massachusetts be laudably
ambitious of not being outdone by Virginians.
The wise and virtuous part of the Fair Sex in Boston and
other Towns, who being at length sensible that by the
consumption of Teas they are supporting the Commissioners
& other Tools of Power, have voluntarily agreed not to give or
receive any further Entertainments of that Kind, until those
Creatures, together with the Boston Standing Army, are
removed, and the Revenue Acts repealed.
May the disgrace which a late venal & corrupt Assembly
has brought upon a Sister Colony, be wiped away by a
Dissolution.
This is pretty plain language, but it could not be strange to the
public ear, for ere this Boston women had been appealed to in the
press upon this same subject.
In the Massachusetts Gazette, as early as November 9, 1767,
these lines show the indignant and revolutionary spirit of the time:
Young ladies in town and those that live round

Let a friend at this season advise you.
Since money’s so scarce and times growing worse,
Strange things may soon hap and surprise you.
First then throw aside your high top knots of pride
Wear none but your own country linen.
Of economy boast. Let your pride be the most
To show cloaths of your own make and spinning.
What if homespun they say is not quite so gay
As brocades, yet be not in a passion,
For when once it is known this is much wore in town,
One and all will cry out ’Tis the fashion.
And as one and all agree that you’ll not married be
To such as will wear London factory
But at first sight refuse, till e’en such you do choose
As encourage our own manufactory.
Soon these frequent appeals, and the influence of the public and
earnest revolt of the Sons of Liberty, resulted in a public compact of
Boston women. It is thus recorded in the Boston press:—
The Boston Evening Post:—
Monday, February 12, 1770.
The following agreement has lately been come into by
upwards of 300 Mistresses of Families in this Town; in which
Number the Ladies of the highest rank and Influence, that
could be waited upon in so short a Time, are included.
Boston, January 31, 1770.
At a time when our invaluable Rights and Privileges are
attacked in an unconstitutional and most alarming Manner,
and as we find we are reproached for not being so ready as
could be desired, to lend our Assistance, we think it our Duty
perfectly to concur with the true Friends of Liberty in all
Measures they have taken to save this abused Country from
Ruin and Slavery. And particularly, we join with the very
respectable Body of Merchants and other Inhabitants of this
Town, who met in Faneuil Hall the 23d of this Instant, in their
Resolutions, totally to abstain from the Use of Tea; And as the
greatest Part of the Revenue arising by Virtue of the late Acts,
is produced from the Duty paid upon Tea, which Revenue is
wholly expended to support the American Board of
Commissioners; We, the Subscribers, do strictly engage, that
we will totally abstain from the Use of that Article, (Sickness
excepted) not only in our respective Families, but that we will
absolutely refuse it, if it should be offered to us upon any
Occasion whatsoever. This Agreement we cheerfully come
into, as we believe the very distressed Situation of our
Country requires it, and we do hereby oblige ourselves
religiously to observe it, till the late Revenue Acts are
repealed.
Massachusetts Gazette, and the Boston Weekly News-Letter:—
February 15, 1770.
We hear that a large Number of the Mistresses of Families,
some of whom are Ladies of the highest Rank, in this Town,
have signed an Agreement against drinking Tea (Bohea it is
supposed, tho’ not specified); they engage not only to abstain
from it in their Families (Sickness excepted) but will absolutely
refuse it, if it should be offered to them upon any Occasion;
This Agreement to be religiously observed till the Revenue
Acts are repealed.
It was natural that, in that hotbed of rebellion, young girls should
not be behind their brothers, fathers, and their mothers in open
avowal of their revolt. Soon the young ladies published this
declaration:—
We, the daughters of those patriots who have and do now
appear for the public interest, and in that principally regard
their posterity—as such, do with pleasure engage with them
in denying ourselves the drinking of foreign tea in hopes to
frustrate a plan which tends to deprive the whole community
of all that is valuable as life.
One dame thus declared her principles and motives in blank
verse:—
Farewell the teaboard with its gaudy equipage

Of cups and saucers, creambucket, sugar tongs,
The pretty tea-chest, also lately stored
With Hyson, Congo and best double-fine.
Full many a joyous moment have I sat by ye
Hearing the girls tattle, the old maids talk scandal,
And the spruce coxcomb laugh at—maybe—nothing.
Though now detestable
Because I am taught (and I believe it true)
Its use will fasten slavish chains upon my country
To reign triumphant in America.
When little Anna Green Winslow bought a hat in February, 1771,
she bought one of “white holland with the feathers sewed on in a
most curious manner, white and unsulleyed as the falling snow. As I
am as we say a daughter of Liberty I chuse to wear as much of our
own manufactory as posible.”
Mercy Warren wrote to John Winthrop, in fine satire upon this
determination of American women to give up all imports from Great
Britain except the necessaries of life, a list of the articles a woman
would deem it imperative to retain:—
An inventory clear
Of all she needs Lamira offers here.
Nor does she fear a rigid Catos frown
When she lays by the rich embroidered gown
And modestly compounds for just enough—
Perhaps some dozen of more slighty stuff.
With lawns and lutestrings, blond and mecklin laces,
Fringes and jewels, fans and tweezer cases,
Gay cloaks and hats of every shape and size,
Scrafs, cardinals and ribbons of all dyes.
With ruffles stamped, and aprons of tambour,
Tippets and handkerchiefs at least three score;
With finest muslins that far India boasts,
And the choice herbage from Chinesan coast.
(But while the fragrant hyson leaf regales
Who’ll wear the home-spun produce of the vales?
For if ’twould save the nation from the curse
Of standing troops—or name a plague still worse,
Few can this choice delicious draught give up,
Though all Medea’s poison fill the cup.)
Add feathers, furs, rich satins and ducapes
And head dresses in pyramidal shapes,
Sideboards of plate and porcelain profuse,
With fifty dittos that the ladies use.
So weak Lamira and her wants are few,
Who can refuse, they’re but the sex’s due.
In youth indeed an antiquated page
Taught us the threatening of a Hebrew page
Gainst wimples, mantles, curls and crisping pins,
But rank not these among our modern sins,
For when our manners are well understood
What in the scale is stomacher or hood?
Tis true we love the courtly mien and air
The pride of dress and all the debonair,
Yet Clara quits the more dressed negligé
And substitutes the careless polanê
Until some fair one from Britannia’s court
Some jaunty dress or newer taste import,
This sweet temptation could not be withstood,
Though for her purchase paid her father’s blood.
After the war had really begun, Mrs. John Adams, writing July 31,
1777, tells of an astonishing action of Boston women, plainly the
result of all these revolutionary tea-notions:—
There is a great scarcity of sugar and coffee, articles which
the female part of the State is very loath to give up, especially
whilst they consider the scarcity occasioned by the merchants
having secreted a large quantity. There had been much rout
and noise in the town for several weeks. Some stores had
been opened by a number of people, and the coffee and
sugar carried into the market and dealt out by pounds. It was
rumored that an eminent stingy wealthy merchant (who is a
bachelor) had a hogshead of coffee in his store which he
refused to sell the committee under six shillings per pound. A
number of females, some say a hundred, some say more,
assembled with a cart and trunks, marched down to the
warehouse and demanded the keys which he refused to
deliver. Upon which one of them seized him by his neck and
tossed him into the cart. Upon his finding no quarter, he
delivered the keys when they tipped up the cart and
discharged him; then opened the warehouse, hoisted out the
coffee themselves, put into the trunks, and drove off. It was
reported that he had personal chastisements among them,
but this I believe was not true. A large concourse of men
stood amazed, silent spectators of the whole transaction.
I suppose these Boston dames thought they might have coffee
since they could not have tea; and, indeed, the relative use of these
two articles in America was much changed by the Revolution. To this
day much more coffee is drunk in America, proportionately, than in
England. We are not a tea-drinking nation.
I don’t know that there were Daughters of Liberty in Philadelphia,
but Philadelphia women were just as patriotic as those of other
towns. One wrote to a British officer as follows:—
I have retrenched every superfluous expense in my table
and family. Tea I have not drunk since last Christmas, nor
have I bought a cap or gown since your defeat at Lexington. I
have learned to knit and am now making stockings of wool for
my servants. In this way do I now throw in my mite for public
good. I know this, that as free I can die but once, but as a
slave I shall not be worthy of life. I have the pleasure to
assure you that these are the sentiments of my sister
Americans.
The women of the South were fired with patriotism; in
Mecklenburgh and Rowan counties, North Carolina, Daughters of
Liberty found another method of spurring patriotism. Young ladies of
the most respectable families banded together, and pledged
themselves not to receive addresses from any recreant suitors who
had not obeyed the country’s call for military service.
There was an historic tea-party also in that town of so much
importance in those days—Edenton, N. C. On October 25, 1774,
fifty-one spirited dames assembled at the residence of Mrs.
Elizabeth King, and passed resolutions commending the action of
the Provincial Congress, and declared also that they would not
conform to “that Pernicious Custom of Drinking Tea or that the
aforesaid Ladys would not promote ye wear of any manufacture from
England,” until the tax was repealed.
The notice of the association is contained in the American
Archives, and runs thus:—
Association Signed by Ladies of Edenton, North Carolina,
Oct. 25, 1774. As we cannot be indifferent on any occasion
that appears to affect the peace and happiness of our country,
and as it has been thought necessary for the publick good to
enter into several particular resolves, by meeting of Members
of Deputies from the whole Province, it is a duty that we owe
not only to our near and dear relations and connections, but
to ourselves who are essentially interested in their welfare, to
do everything as far as lies in our power to testify our sincere
adherence to the same, and we do therefore accordingly
subscribe this paper as a witness of our fixed intentions and
solemn determination to do so. Signed by fifty one ladies.
It is a good example of the strange notions which some historians
have of the slight value of circumstantial evidence in history, that the
names of these fifty-one ladies have not been preserved. A few,
however, are known. The president was Mrs. Penelope Barker, who
was thrice a widow, of husbands Hodgson, Crumm, and Barker. She
was high-spirited, and from her varied matrimonial experiences knew
that it was needless to be afraid of any man; so when British soldiers
invaded her stables to seize her carriage horses, she snatched the
sword of one of her husbands from the wall, with a single blow
severed the reins in the British officer’s hands, and drove her horses
back into the stables, and kept them too.
The fame of this Southern tea-party reached England, for Arthur
Iredell wrote (with the usual masculine jocularity upon feminine
enterprises) thus, on January 31, 1775, from London to his patriot
brother, James Iredell:—
I see by the newspapers the Edenton ladies have
signalized themselves by their protest against tea-drinking.
The name of Johnston I see among others; are any of my
sister’s relations patriotic heroines? Is there a female
Congress at Edenton too? I hope not, for we Englishmen are
afraid of the male Congress, but if the ladies who have ever,
since the Amazonian era, been esteemed the most
formidable enemies, if they, I say, should attack us, the most
fatal consequence is to be dreaded. So dextrous in the
handling of a dart, each wound they give is mortal; whilst we,
so unhappily formed by Nature, the more we strive to conquer
them the more are conquered! The Edenton ladies, conscious
I suppose of this superiority on their side, by former
experience, are willing, I imagine, to crush us into atoms by
their omnipotency; the only security on our side to prevent the
impending ruin that I can perceive is the probability that there
are few places in America which possess so much female
artillery as in Edenton.
Another indication of the fame of the Edenton tea-party is adduced
by Dr. Richard Dillard in his interesting magazine paper thereon. It
was rendered more public by a caricature, printed in London, a
mezzotint, entitled “A Society of Patriotic Ladies at Edenton in North
Carolina.” One lady with a gavel is evidently a man in woman’s
clothing, and is probably intended for the hated Lord North; other
figures are pouring the tea out of caddies, others are writing. This
caricature may have been brought forth in derision of an interesting
tea-party picture which still exists, and is in North Carolina, after
some strange vicissitudes in a foreign land. It is painted on glass,
and the various figures are doubtless portraits of the Edenton ladies.
It is difficult to-day to be wholly sensible of all that these Liberty
Bands meant to the women of the day. There were not, at that time,
the associations of women for concerted charitable and philanthropic
work which are so universal now. There were few established and
organized assemblies of women for church work (there had been
some praying-meetings in Whitefield’s day), and the very thought of
a woman’s society for any other than religious purposes must have
been in itself revolutionary. And we scarcely appreciate all it meant
for them to abandon the use of tea; for tea-drinking in that day meant
far more to women than it does now. Substitutes for the taxed and
abandoned exotic herb were eagerly sought and speedily offered.
Liberty Tea, Labrador Tea, and Yeopon were the most universally
accepted, though seventeen different herbs and beans were named
by one author; and patriotic prophecies were made that their use
would wholly outlive that of the Oriental drink, even could the latter
be freely obtained. A century has proved the value of these
prophecies.
Liberty Tea was the most popular of these Revolutionary
substitutes. It sold for sixpence a pound. It was made from the four-
leaved loose-strife, a common-growing herb. It was pulled up whole
like flax, its stalks were stripped of the leaves and then boiled. The
leaves were put in a kettle with the liquor from the stalks and again
boiled. Then the leaves were dried in an oven. Sage and rib-wort,
strawberry leaves and currant leaves, made a shift to serve as tea.
Hyperion or Labrador Tea, much vaunted, was only raspberry
leaves, but was not such a wholly odious beverage. It was loudly
praised in the patriotic public press:—
The use of Hyperion or Labrador tea is every day coming
into vogue among people of all ranks. The virtues of the plant
or shrub from which this delicate Tea is gathered were first
discovered by the Aborigines, and from them the Canadians
learned them. Before the cession of Canada to Great Britain
we knew little or nothing of this most excellent herb, but since
we have been taught to find it growing all over hill and dale
between the Lat. 40 and 60. It is found all over New England
in great plenty and that of best quality, particularly on the
banks of the Penobscot, Kennebec, Nichewannock and
Merrimac.
CHAPTER XI.
A REVOLUTIONARY HOUSEWIFE.
We do not need to make a composite picture of the housewife of

Revolutionary days, for a very distinct account has been preserved
of one in the quaint pages of the Remembrancer or diary of
Christopher Marshall, a well-to-do Quaker of Philadelphia, who was
one of the Committee of Observation of that city during the
Revolutionary War. After many entries through the year 1778, which
incidentally show the many cares of his faithful wife, and her
fulfilment of these cares, the fortunate husband thus bursts forth in
her praise:—
As I have in this memorandum taken scarcely any notice of
my wife’s employments, it might appear as if her
engagements were very trifling; the which is not the case but
the reverse. And to do her justice which her services
deserved, by entering them minutely, would take up most of
my time, for this genuine reason, how that from early in the
morning till late at night she is constantly employed in the
affairs of the family, which for four months has been very
large; for besides the addition to our family in the house, it is a
constant resort of comers and goers which seldom go away
with dry lips and hungry bellies. This calls for her constant
attendance, not only to provide, but also to attend at getting
prepared in the kitchen, baking our bread and pies, meat &c.
and also the table. Her cleanliness about the house, her
attendance in the orchard, cutting and drying apples of which
several bushels have been procured; add to which her
making of cider without tools, for the constant drink of the
family, her seeing all our washing done, and her fine clothes
and my shirts, the which are all smoothed by her; add to this,
her making of twenty large cheeses, and that from one cow,
and daily using with milk and cream, besides her sewing,
knitting &c. Thus she looketh well to the ways of her
household, and eateth not the bread of idleness; yea she also
stretcheth out her hand, and she reacheth forth her hand to
her needy friends and neighbors. I think she has not been
above four times since her residence here to visit her
neighbors; nor through mercy has she been sick for any time,
but has at all times been ready in any affliction to me or my
family as a faithful nurse and attendant both day and night.
Such laudatory references to the goodwife as these abound
through the Remembrancer.
My tender wife keeps busily engaged and looks upon every
Philadelphian who comes to us as a person suffering in a
righteous cause; and entitled to partake of her hospitality
which she administers with her labor and attendance with
great freedom and alacrity....
My dear wife meets little respite all the day, the proverb
being verified, that Woman’s Work is never done.
I owe my health to the vigilance, industry and care of my
wife who really has been and is a blessing unto me. For the
constant assiduity and press of her daily and painful labor in
the kitchen, the Great Lord of the Household will reward her in
due time.
It seems that so generous and noble a woman should have had a
reward in this world, as well as the next, for, besides her kitchen
duties, she was a “nonsuch gardner, working bravely in her garden,”
and a first class butter-maker, who constantly supplied her poor
neighbors with milk, and yet always had cream to spare for her dairy.
Far be it from me to cast even the slightest reflection, to express
the vaguest doubt, as to the industry, energy, and application of so
pious, so estimable an old gentleman as Mr. Marshall, but he was,
as he says, “easily tired”—“the little I do tires and fatigues me”—“the
grasshopper seems a burden.” So, even to our prosaic and
somewhat emancipated nineteenth century notions as to women’s
rights and their assumption of men’s duties, it does appear that so
patient, industrious, and overworked a consort might have been
spared some of the burdensome duties which devolved upon her,
and which are popularly supposed not to belong to the distaff side of
the house. An elderly milk-man might have occasionally milked the
cow for that elderly weary milkmaid. And it does seem just a little
strange that a hearty old fellow, who could eat gammons and drink
punch at every occasion of sober enjoyment and innocent revelry to
which he was invited, should let his aged spouse rise at daybreak
and go to the wharves to buy loads of wood from the bargemen; and
also complacently record that the horse would have died had not the
ever-energetic wife gone out and by dint of hard work and good
management succeeded in buying in the barren city a load of hay for
provender. However, he never fails to do her justice in
commendatory words in the pages of his Remembrancer, thus
proving himself more thoughtful than that Yankee husband who said
to a neighbor that his wife was such a good worker and a good cook,
and so pleasant and kept everything so neat and nice around the
house, that sometimes it seemed as if he couldn’t help telling her so.
One of the important housewifely cares of Philadelphia women
was their marketing, and Madam Marshall was faithful in this duty
also. We find her attending market as early as four o’clock upon a
winter’s morning. In 1690, there were two market days weekly in
Philadelphia, and nearly all the early writers note the attendance
thereat of the ladies residing in the town. In 1744, these markets
were held on Tuesday and Friday. William Black, a travelling
Virginian, wrote that year with admiration of this custom:—
I got to the market by 7, and had no small Satisfaction in
seeing the pretty Creatures, the Young Ladies, traversing the
place from Stall to Stall where they could make the best
Market, some with their maid behind them with a Basket to
carry home the Purchase, others that were design’d to buy
but trifles, as a little fresh Butter, a Dish of Green Peas or the
like, had Good Nature & Humility enough to be their own
Porters. I have so much regard for the fair Sex that I imagin’d
like the Woman of the Holy Writ some charm in touching even
the Hem of their Garments. After I made my Market, which
was one pennyworth of Whey and a Nosegay, I disengag’d
myself.
It would appear also that a simple and appropriate garment was
donned for this homely occupation. We find Sarah Eve and others
writing of wearing a “market cloke.”
It is with a keen thrill of sympathy that we read of all the torment
that Mistress Marshall, that household saint, had to endure in the
domestic service rendered to her—or perhaps I should say through
the lack of service in her home. A special thorn in the flesh was one
Poll, a bound girl. On September 13, 1775, Mr. Marshall wrote:—
After my wife came from market (she went past 5) she
ordered her girl Poll to carry the basket with some
necessaries to the place, as she was coming after her, they
intending to iron the clothes. Poll accordingly went, set down
the basket, came back, went and dressed herself all clean,
short calico gown, and said she was going to school; but
presently after the negro woman Dinah came to look for her,
her mistress having mistrusted she had a mind to play truant.
This was about nine, but madam took her walk, but where—
she is not come back to tell.
Sept. 16. I arose before six as I was much concern’d to see
my wife so afflicted as before on the bad conduct of her girl
Poll who is not yet returned, but is skulking and running about
town. This I understand was the practice of her mother who
for many years before her death was a constant plague to my
wife, and who left her this girl as a legacy, and who by report
as well as by own knowledge, for almost three years has
always been so down to this time. About eight, word was
brought that Poll was just taken by Sister Lynn near the
market, and brought to their house. A messenger was
immediately dispatched for her, as she could not be found
before, though a number of times they had been hunting her.
As the years went on, Poll kept taking what he called “cruises,”
“driving strokes of impudence,” visiting friends, strolling around the
streets, faring up and down the country, and he patiently writes:—
This night our girl was brought home. I suppose she was
hunted out, as it is called, and found by Ruth on the Passyunk
Road. Her mistress was delighted upon her return, but I know
of nobody else in house or out. I have nothing to say in the
affair, as I know of nothing that would distress my wife so
much as for me to refuse or forbid her being taken into the
house.
(A short time after) I arose by four as my wife had been up
sometime at work cleaning house, and as she could not rest
on account of Polls not being yet return’d. The girls frolics
always afflict her mistress, so that to me its plain if she does
not mend, or her mistress grieve less for her, that it will
shorten Mrs Marshalls days considerably; besides our house
wears quite a different face when Miss Poll is in it (although
all the good she does is not worth half the salt she eats.) As
her presence gives pleasure to her mistress, this gives joy to
all the house, so that in fact she is the cause of peace or
uneasiness in the home.
It is with a feeling of malicious satisfaction that we read at last of
the jaded, harassed, and conscientious wife going away for a visit,
and know that the man of the house will have to encounter and
adjust domestic problems as best he may. No sooner had the
mistress gone than Poll promptly departed also on a vacation. As
scores of times before, Mr. Marshall searched for her, and retrieved
her (when she was ready to come), and she behaved exceeding well
for a day, only, when rested, to again make a flitting. He writes on the
23d:—
I roused Charles up at daylight. Found Miss Poll in the
straw house. She came into the kitchen and talked away that
she could not go out at night but she must be locked out. If
that’s the case she told them she would pack up her clothes
and go quite away; that she would not be so served as her
Mistress did not hinder her staying out when she pleased, and
the kitchen door to be opened for her when she came home
and knocked. The negro woman told me as well as she could
what she said. I then went and picked up her clothes that I
could find. I asked her how she could behave so to me when I
had conducted myself so easy towards her even so as to
suffer her to sit at table and eat with me. This had no effect
upon her. She rather inclined to think that she had not
offended and had done nothing but what her mistress
indulged her in. I told her before Betty that it was not worth my
while to lick her though she really deserved it for her present
impudence; but to remember I had taken all her clothes I
could find except what she had on, which I intended to keep;
that if she went away Charles with the horse should follow her
and bring her back and that I would send a bellman around
the borough of Lancaster to cry her as a runaway servant,
wicked girl, with a reward for apprehending her.
The fatuous simplicity of Quaker Marshall’s reproofs, the futility of
his threats, the absurd failure of his masculine methods, received
immediate illustration—as might be expected, by Miss Poll promptly
running away that very night. Again he writes:—
Charles arose near daybreak and I soon after, in order to
try to find my nightly and daily plague, as she took a walk
again last night. Charles found her. We turned her upstairs to
refresh herself with sleep....
(Two days later) After breakfast let our Poll downstairs
where she has been kept since her last frolic. Fastened her
up again at night. I think my old enemy Satan is much
concerned in the conduct and behavior of that unfortunate
girl. He knows her actions give me much anxiety and indeed
at times raise my anger so I have said what should have been
avoided, but I hope for the future to be more upon my guard
and thus frustrate him in his attempts.
With what joy did the masculine housekeeper and steward greet
the return of his capable wife, and resign his position as turnkey!
Poll, upon liberation from restraint, flew swiftly away like any other
bird from its cage.
Notwithstanding such heavy weather overhead and
exceeding dirty under foot our Poll after breakfast went to see
the soldiers that came as prisoners belonging to Burgoynes
army. Our trull returned this morning. Her mistress gave her a
good sound whipping. This latter was a variety.
And so the unequal fight went on; Poll calmly breaking down a
portion of the fence that she might decamp more promptly, and
return unheralded. She does not seem to have been vicious, but
simply triumphantly lawless and fond of gadding. I cannot always
blame her. I am sure I should have wanted to go to see the soldier-
prisoners of Burgoyne’s army brought into town. The last glimpse of
her we have is with “her head dressed in tiptop fashion,” rolling off in
a coach to Yorktown with Sam Morris’s son, and not even saying
good-by to her vanquished master.
Mr. Marshall was not the only Philadelphian to be thus afflicted; we
find one of his neighbors, Jacob Hiltzheimer, dealing a more
summary way with a refractory maid-servant. Shortly after noting in
the pages of his diary that “our maid Rosina was impertinent to her
mistress,” we find this good citizen taking the saucy young
redemptioner before the squire, who summarily ordered her to the
workhouse. After remaining a month in that confinement, Rosina
boldly answered no, when asked if she would go back to her master
and behave as she ought, and she was promptly remanded. But she
soon repented, and was released. Her master paid for her board and
lodging while under detention, and quickly sold her for £20 for her
remaining term of service.
With the flight of the Marshalls’ sorry Poll, the sorrows and trials of
this good Quaker household with regard to what Raleigh calls
“domesticals” were not at an end. As the “creatures” and the orchard
and garden needed such constant attention, a man-servant was
engaged—one Antony—a character worthy of Shakespeare’s
comedies. Soon we find the master writing:—
I arose past seven and had our gentleman to call down
stairs. I spoke to him about his not serving the cows. He at
once began about his way being all right, &c. I set about
serving our family and let him, as in common, do as he
pleases. I think I have hired a plague to my spirit. Yet he is still
the same Antony—he says—complaisant, careful, cheerful,
industrious.
Then Antony grew noisy and talkative, so abusive at last that he
had to be put out in the yard, where he railed and talked till midnight,
to the annoyance of the neighbors and the mortification of his
mistress; for he protested incessantly and noisily that all he wished
was to leave in peace and quiet, which he was not permitted to do.
Then, and repeatedly, his master told him to leave, but the servant
had no other home, and might starve in the war-desolated town; so
after half-promises he was allowed by these tender folk to stay on.
Soon he had another “tantrum,” and the astounded Quaker writes:—
He rages terribly uttering the most out of the way wicked
expressions yet not down-right swearing. Mamma says it is
cursing in the Popish way....
What this Popish swearing could have been arouses my curiosity;
I suspect it was a kind of “dog-latin.” Antony constantly indulged in it,
to the horror and sorrow of the pious Marshalls. And the amusing,
the fairly comic side of all this is that Antony was a preacher, a
prophet in the land, and constantly held forth in meeting to sinners
around him. We read of him:—
Antony went to Quakers meeting today where he preached;
although he was requested to desist, so that by consent they
broke up the meeting sooner than they would have done....
Mamma went to meeting where Antony spoke and was
forbid. He appeared to be most consummately bold and
ignorant in his speaking there. And about the house I am
obliged in a stern manner at times to order him not to say one
word more....
This afternoon Antony preached at the English Presbyterian
meeting. It is said that the hearers laughed at him but he was
highly pleased with himself.
Antony preached at meeting. I kept engaged helping to
cook the pot against master came home. He comes and goes
as he pleases.
I don’t know when to pity poor Dame Marshall the most, with
Antony railing in the yard and disturbing the peace of the neighbors;
or Antony cursing in a Popish manner through the house; or Antony
shamming sick and moaning by the fireside; or Antony violently
preaching when she had gone to the quiet Quaker meeting for an
hour of peace and rest.
This “runnagate rascal” was as elusive, as tricky, as malicious as a
gnome; whenever he was reproved, he always contrived to invent a
new method of annoyance in revenge. When chidden for not feeding
the horse, he at once stripped the leaves off the growing cabbages,
cut off the carrot heads, and pulled up the potatoes, and pretended
and protested he did it all solely to benefit them, and thus do good to
his master. When asked to milk the cow, he promptly left the
Marshall domicile for a whole day.
Sent Antony in the orchard to watch the boys. As I was
doubtful sometime whether if any came for apples Antony
would prevent, I took a walk to the back fence, made a noise
by pounding as if I would break the fence, with other noise.
This convinced me Antony sat in his chair. He took no notice
till my wife and old Rachel came to him, roused him, and
scolded him for his neglect. His answer was that he thought it
his duty to be still and not disturb them, as by so doing he
should have peace in heaven and a blessing would ever
attend him.
This was certainly the most sanctimonious excuse for laziness that
was ever invented; and on the following day Antony supplemented
his tergiversation by giving away all Mr. Marshall’s ripe apples
through the fence to passers-by—neighbors, boys, soldiers, and
prisoners. There may have been method in this orchard madness,
for Antony loathed apple-pie, a frequent comestible in the Marshall
domicile, and often refused to drink cider, and grumbling made toast-
tea instead. In a triumph of euphuistic indignation, Mr. Marshall thus
records the dietetic vagaries of the “most lazy impertinent talking
lying fellow any family was ever troubled with:”
When we have no fresh broth he wants some; when we
have it he cant sup it. When we have lean of bacon he wants
the fat; when the fat he cant eat it without spreading salt over
it as without it its too heavy for his stomach. If new milk he
cant eat it till its sour, it curdles on his stomach; when sour or
bonnyclabber it gives him the stomach-ache. Give him tea he
doesn’t like such slop, its not fit for working men; if he hasn’t it
when he asks for it he’s not well used. Give him apple pie
above once for some days, its not suitable for him it makes
him sick. If the negro woman makes his bed, she dont make it
right; if she dont make it she’s a lazy black jade, &c.
In revenge upon the negro woman Dinah for not making his bed to
suit his notion, he pretended to have had a dream about her, which
he interpreted to such telling effect that she thought Satan was on
his swift way to secure her, and fled the house in superstitious fright,
in petticoat and shift, and was captured three miles out of town. On
her return, Antony outdid himself with “all the vile ribaldry, papist
swearing, incoherent scurrilous language, that imperious pride,
vanity, and folly could invent or express”—and then went off to
meeting to preach and pray. Well might the Quaker say with Juvenal,
“The tongue is the worst part of a bad servant.” At last, exasperated
beyond measure, his patient master vowed, “Antony, I will give thee
a good whipping,” and he could do it, for he had “pacified himself
with sundry stripes of the cowskin” on Dinah, the negro, when she, in
emulation of Antony, was impertinent to her mistress.
The threat of a whipping brought on Antony a “fit of stillness” which
descended like a blessing on the exhausted house. But “the devil is
sooner raised than laid;” anon Antony was in his old lunes again, and
the peace was broken by a fresh outburst of laziness, indifference,
and abuse, in which we must leave this afflicted household, for at
that date the Remembrancer abruptly closes.
The only truly good service rendered to those much tried souls
was by a negro woman, Dinah, who, too good for this earth, died;

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Bacteriophages Practical Applications

for Nature s Biocontrol 1st Edition

Cottage Industry of Biocontrol Agents and Their

Concise Illustrated Dictionary of Biocontrol Terms 1st

Bacteriophages Methods and Protocols Ebenezer Tumban

Timeless Nature s Formula for Health and Longevity 1st

Artificial Intelligence for Marketing Practical

Predictive Modeling with SAS Enterprise Miner Practical

Ecological and Practical Applications for Sustainable

Practical Entity Framework: Database Access for

ISBN 978-3-319-54050-4 ISBN 978-3-319-54051-1 (eBook)

© Springer International Publishing AG 2017

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Sabah A.A. Jassim

We ﬁnd ourselves in the twenty-ﬁrst century with a world of disenchantment, a

bacteria from sensitive environments. Bacteriophages can be effective in decon-

Windsor, ON, Canada Sabah A.A. Jassim

2.7 Phage, AMR and Virulence Factors in Bacteria Sharing . . . . . . . 31

4.2.2 Livestock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

6.2 Cyanophage Biocontrol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

Professor Sabah A.A. Jassim Adjunct Professor, Civil and Environmental

phages as a novel, environmentally friendly biocontrol, particularly in agricultural

Mr. Richard G. Limoges currently a businessman, is operating two successful

AAP American Academy of Pediatrics

QRA Quantitative Risk Assessment

Keywords Bacteriophage Phage design Phage breeding Phage reprogram-

© Springer International Publishing AG 2017 1

environmentally-driven and so mimics natural selection or evolution of the phage

1. PBS: Dissolve one tablet in 100 ml of a 1X solution. 1X PBS solution contains

1.2.4 Preparation of FeSO4.7H2O Solution

1. First freshly prepare solution (0.01%) of FeSO4.7H2O in Lambda-buffer, pH 6.3.

1.2.5 Preparation of 13% Pomegranate Rind Extract (PRE)

1.2.6 Antiviral (Anti-phage) Agent (Jassim et al. 1995)

Immediately prior to use, transfer 3.3 ml of 13% PRE added to 7 ml of freshly

Carry out all procedures at room temperature unless otherwise stated.

1.3.1 Bacterial Stock Culture

1. Prior to procedure, a stock culture of the phage target bacteria should be

1.3.2 Phage Hunting/Isolation Techniques

1. Collect crude specimens of approximately 50 g of environmental dirt, sewage

1.3.3 Testing for the Presence of Crude Wild Phages

1. Prepare bacterial lawns of target bacterial isolates or reference bacterial strains

1.3.4 Production of the Transient Phage Stock

1.3.5 Optimization of the Phages Lytic Characteristics

1.3.5.1 Plaque-Based Optimization

1.3.5.2 Phage Enhancement of Lytic Activity

1.3.6 Vertical Optimization for Phage-Host Interaction

Fig. 1.2 Principle of vertical optimization for phage-host interaction

1.3.6.1 Phage Mass-Action Biokinetics to Select the Elite Phages

3. At appropriate contact time 2, 5, 7, 10, and 15 min transfer 20 ll from

1.3.6.2 Propagation of the Elite Phages

1.3.6.3 Phage Titer

1. Make 10-fold serial dilutions of the selected phage in lambda buffer.

1.3.6.4 Phage Titer Calculations

ð# of plaquesÞ ðdilution factorÞ ð1000 ll=mlÞ

ð35 pfuÞ ð107 Þ ð1000 ll=mlÞ

1.3.6.5 Improve Phage Adsorption ‘Attachment’ Time

1.3.6.6 Infective Ratio (IR)

# of phage particles in the pre burst era at a given dilution

Young ladies in town and those that live round

Farewell the teaboard with its gaudy equipage

We do not need to make a composite picture of the housewife of

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