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Methods in
Molecular Biology 1693
Joana Azeredo
Sanna Sillankorva Editors
Bacteriophage
Therapy
From Lab to Clinical Practice
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Bacteriophage Therapy
From Lab to Clinical Practice
Edited by
Joana Azeredo
LIBRO-Laboratory of Research in Bioﬁlms Rosário Oliveira, CEB – Centre of Biological Engineering,
University of Minho, Braga, Portugal
Sanna Sillankorva
CEB – Centre of Biological Engineering, University of Minho, Braga, Portugal
Editors
Joana Azeredo Sanna Sillankorva
LIBRO-Laboratory of Research CEB – Centre of Biological Engineering
in Biofilms Rosário Oliveira University of Minho
CEB – Centre of Biological Engineering Braga, Portugal
University of Minho
Braga, Portugal
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-7394-1 ISBN 978-1-4939-7395-8 (eBook)
DOI 10.1007/978-1-4939-7395-8
Library of Congress Control Number: 2017956899
© Springer Science+Business Media LLC 2018

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Preface
This year we are celebrating the 100th anniversary of bacteriophage discovery. Bacterio-
phage therapy has had glorious and unfortunate periods. Since 1917, bacteriophages were
extensively and intensively used as antimicrobial agents to control bacterial infections. In the
early part of the century many big pharmaceutical companies produced several bacterio-
phage products for different applications. The discovery of antibiotics allied to the indis-
criminate use of bacteriophages to treat every sort of infection, even nonbacterial, is
identified as one of the major reasons that led to the abandonment of bacteriophage therapy.
Antibiotics have now been used for approximately 70 years and have reduced illnesses and
deaths caused by infectious diseases. However, the emergence of antibiotic resistant bacteria
is a major health concern. Each year millions of people become infected with multiple-drug
resistant bacteria, which consequently leads to numerous deaths. On this account, there is a
revival of interest in bacteriophage as therapeutic agents to control infectious diseases.
Despite this renewed interest, there is not yet any product broadly available for human
application. There are however a few exceptions where bacteriophages can be applied to
human patients as a last resort under the guidelines of the Helsinki treaty and as a therapeu-
tic option in a few European countries. Accordingly, it is important to bring to medical
application the products that are being developed at the lab. Bacteriophage Therapy: From
Lab to Clinical Practice focuses on the methodology of product development and applica-
tion in clinical context to ensure efficacy, safety, and regulatory compliance.
This book has 21 chapters which have been subgrouped by dividing the experimental
approaches suitable for isolating and characterizing bacteriophages to formulating bacterio-
phage medicinal products and clinical application. Regulatory compliance and safety aspects
of bacteriophage therapy are also addressed in the book.
We would like to express our gratitude to all contributing authors whose expertise in the
field is highly recognized for their commitment to this book by sharing their knowledge in a
simple and comprehensive way to guide bacteriophage researchers throughout the develop-
ment of a product for medical application. This book also targets a broader audience ranging
from clinicians, pharmaceutics, regulatory authorities, and stakeholders.
Braga, Portugal Joana Azeredo

Sanna Sillankorva
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I ISOLATION OF BACTERIOPHAGES

1 Isolation of Bacteriophages for Fastidious Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Shigenobu Matsuzaki, Jumpei Uchiyama, Iyo Takemura-Uchiyama,
Takako Ujihara, and Masanori Daibata
2 Isolation of Bacteriophages of the Anaerobic Bacteria Bacteroides . . . . . . . . . . . . . 11
Cristina Garcı́a-Aljaro, Maite Muniesa, and Juan Jofre
3 Isolation of Bacteriophages for Clinically Relevant Bacteria. . . . . . . . . . . . . . . . . . . 23
Sanna Sillankorva
PART II CHARACTERIZATION OF BACTERIOPHAGES
4 In Vitro Activity of Bacteriophages Against Planktonic and

Biofilm Populations Assessed by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Diana P. Pires and Luı́s D.R. Melo
5 Observation of Bacteriophage Ultrastructure by Cryo-electron Microscopy . . . . 43
Ana Cuervo and José L. Carrascosa
6 Bacteriophage Taxonomy: An Evolving Discipline . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Igor Tolstoy, Andrew M. Kropinski, and J. Rodney Brister
PART III BACTERIOPHAGE SELECTION AND COCKTAIL FORMULATION
7 Determination of the Bacteriophage Host Range:

Culture-Based Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Andrey V. Letarov and Eugene E. Kulikov
8 Recovery and Characterization of Bacteria Resisting Infection
by Lytic Bacteriophage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Libera Latino and Christine Pourcel
9 Guidelines to Compose an Ideal Bacteriophage Cocktail . . . . . . . . . . . . . . . . . . . . . 99
Maia Merabishvili, Jean-Paul Pirnay, and Daniel De Vos
PART IV BIODISTRIBUTION, HOST INTERACTION AND CLINICAL APPLICATION

10 Interaction of Bacteriophages with Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . 113
Zuzanna Kaźmierczak and Krystyna Da˛browska
11 In Vivo Bacteriophage Biodistribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Nicolas Dufour, Raphaëlle Delattre, and Laurent Debarbieux
vii
viii Contents
12 Interaction of Bacteriophages with the Immune System:

Induction of Bacteriophage-Specific Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Krystyna Da˛browska
13 Bacteriophage Treatment of Infected Diabetic Foot Ulcers . . . . . . . . . . . . . . . . . . 151
Vera V. Morozova, Yulia N. Kozlova, Denis A. Ganichev,
and Nina V. Tikunova
14 Compassionate Use of Bacteriophage Therapy for Foot Ulcer
Treatment as an Effective Step for Moving Toward Clinical Trials. . . . . . . . . . . . . 159
Randolph Fish, Elizabeth Kutter, Gordon Wheat, Bob Blasdel,
Mzia Kutateladze, and Sarah Kuhl
PART V PRODUCTION, PURIFICATION AND BACTERIOPHAGE STORAGE
15 Bacteriophage Production in Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

Maryam Agboluaje and Dominic Sauvageau
16 Computational Modeling of Bacteriophage Production for
Process Optimization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Konrad Krysiak-Baltyn, Gregory J.O. Martin, and Sally L. Gras
17 Methods for Bacteriophage Preservation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Małgorzata B. Łobocka, Aleksandra Głowacka, and Piotr Golec
PART VI SAFETY AND REGULATION
18 Bacteriophage Production in Compliance with

Regulatory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Jean-Paul Pirnay, Maia Merabishvili, Hilde Van Raemdonck,
Daniel De Vos, and Gilbert Verbeken
19 Guidelines for Bacteriophage Product Certification . . . . . . . . . . . . . . . . . . . . . . . . . 253
Alan Fauconnier
PART VII NEW PHAGE THERAPY APPROACHES
20 Nano/Micro Formulations for Bacteriophage Delivery . . . . . . . . . . . . . . . . . . . . . . 271

Pilar Cortés, Mary Cano-Sarabia, Joan Colom, Jennifer Otero,
Daniel Maspoch, and Montserrat Llagostera
21 Synthetic Biology to Engineer Bacteriophage Genomes . . . . . . . . . . . . . . . . . . . . . 285
Ana Rita Costa, Catarina Milho, Joana Azeredo,
and Diana Priscila Pires
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Contributors
MARYAM AGBOLUAJE Chemical and Materials Engineering, University of Alberta,

Edmonton, AB, Canada
JOANA AZEREDO LIBRO - Laboratory of Research in Biofilms Rosário Oliveira, CEB –
Centre of Biological Engineering, University of Minho, Braga, Portugal
BOB BLASDEL Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg, Belgium
J. RODNEY BRISTER National Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, Bethesda, MD, USA
MARY CANO-SARABIA Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC
and The Barcelona Institute of Science and Technology, Barcelona, Spain
JOSÉ L. CARRASCOSA Department of Structure of Macromolecules, Centro Nacional de
Biotecnologı́a, Madrid, Spain
JOAN COLOM School of Veterinary Medicine and Science, University of Nottingham,
Nottingham, UK
PILAR CORTÉS Departament de Genètica i Microbiologia, Universitat Autònoma de
Barcelona, Cerdanyola del Vallès, Spain
ANA RITA COSTA LIBRO - Laboratory of Research in Biofilms Rosário Oliveira, CEB –
ANA CUERVO Department of Structure of Macromolecules, Centro Nacional de
Biotecnologı́a, Madrid, Spain
KRYSTYNA DA˛BROWSKA Institute of Immunology and Experimental Therapy, Polish Academy
of Sciences, Wrocław, Poland
MASANORI DAIBATA Department of Microbiology and Infection, Kochi Medical School, Kochi
University, Nankoku, Kochi, Japan
LAURENT DEBARBIEUX Department of Microbiology, Molecular Biology of Gene in
Extremophiles, Institut Pasteur, Paris, France
RAPHAËLLE DELATTRE Department of Microbiology, Molecular Biology of Gene in
Extremophiles, Institut Pasteur, Paris, France; INSERM, IAME, UMR 1137, Paris,
France; Service d’Anesthésie-Réanimation, AP-HP, Hôpital Beaujon, Clichy, France
NICOLAS DUFOUR Department of Microbiology, Molecular Biology of Gene in Extremophiles,
Institut Pasteur, Paris, France; Service de Réanimation Médico-Chirurgicale, Centre
Hospitalier René Dubos, Pontoise, France; INSERM, IAME, UMR 1137, Paris, France
ALAN FAUCONNIER Federal Agency for Medicines and Health Products, Brussels, Belgium;
Culture in vivo ASBL, Nivelles, Belgium
RANDOLPH FISH PhageBiotics Research Foundation, Gray’s Harbor Community Hospital,
Aberdeen, WA, USA; Wound Centers at St Joseph’s Medical Center, Tacoma, WA, USA
DENIS A. GANICHEV Railway Clinical Hospital, Novosibirsk, Russian Federation
CRISTINA GARCÍA-ALJARO Department of Microbiology, University of Barcelona, Barcelona,
Spain
ALEKSANDRA GŁOWACKA Department of Microbial Biochemistry, Institute of Biochemistry
and Biophysics of the Polish Academy of Sciences, Warsaw, Poland
PIOTR GOLEC Laboratory of Molecular Biology (affiliated with the University of Gdańsk),
Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Gdańsk, Poland
ix
x Contributors
SALLY L. GRAS The Bio21 Molecular Science and Biotechnology Institute, The University of
Melbourne, Parkville, VIC, Australia; The Department of Chemical Engineering, The
University of Melbourne, Parkville, VIC, Australia
JUAN JOFRE Department of Microbiology, University of Barcelona, Barcelona, Spain
ZUZANNA KAŹMIERCZAK Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, Wrocław, Poland
YULIA N. KOZLOVA Laboratory of Molecular Microbiology, Institute of Chemical Biology and
Fundamental Medicine SB RAS, Novosibirsk, Russian Federation
ANDREW M. KROPINSKI Department of Food Science, University of Guelph, Guelph, ON,
Canada; Department of Molecular & Cellular Biology, University of Guelph, Guelph, ON,
Canada; Department of Pathobiology, University of Guelph, Guelph, ON, Canada
KONRAD KRYSIAK-BALTYN The Bio21 Molecular Science and Biotechnology Institute, The
University of Melbourne, Parkville, VIC, Australia; The Department of Chemical
Engineering, The University of Melbourne, Parkville, VIC, Australia
SARAH KUHL VA Northern California, Martinez, CA, USA; University of California, San
Francisco, CA, USA
EUGENE E. KULIKOV Winogradsky Institute of Microbiology RC Biotechnology RAS, Moscow,
Russia; Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia
MZIA KUTATELADZE George Eliava Institute of Bacteriophages, Microbiology and Virology –
3, Tbilisi, GA, USA
ELIZABETH KUTTER PhageBiotics Research Foundation, Gray’s Harbor Community
Hospital, Aberdeen, WA, USA; Bacteriophage Lab, The Evergreen State College, Olympia,
WA, USA
LIBERA LATINO Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.
Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette Cedex, France
ANDREY V. LETAROV Winogradsky Institute of Microbiology RC Biotechnology RAS, Moscow,
Russia; Lomonosov Moscow State University, Moscow, Russia; Moscow Institute of Physics
and Technology, Dolgoprudny, Moscow Region, Russia
MONTSERRAT LLAGOSTERA Departament de Genètica i Microbiologia, Universitat
Autònoma de Barcelona, Cerdanyola del Vallès, Spain
MAŁGORZATA B. ŁOBOCKA Department of Microbial Biochemistry, Institute of Biochemistry
and Biophysics of the Polish Academy of Sciences, Warsaw, Poland; Faculty of Agriculture
and Biology, Autonomous Department of Microbial Biology, Warsaw University of Life
Sciences – SGGW, Warsaw, Poland
GREGORY J.O. MARTIN The Department of Chemical Engineering, The University of
Melbourne, Parkville, VIC, Australia
DANIEL MASPOCH Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC
and The Barcelona Institute of Science and Technology, Barcelona, Spain; ICREA,
Barcelona, Spain
SHIGENOBU MATSUZAKI Department of Microbiology and Infection, Kochi Medical School,
Kochi University, Nankoku, Kochi, Japan
LUÍS D.R. MELO LIBRO – Laboratory of Research in Biofilms Rosário Oliveira, CEB –
MAIA MERABISHVILI Laboratory for Molecular and Cellular Technology, Queen Astrid
Military Hospital, Brussels, Belgium
CATARINA MILHO LIBRO - Laboratory of Research in Biofilms Rosário Oliveira, CEB –
Contributors xi
VERA V. MOROZOVA Laboratory of Molecular Microbiology, Institute of Chemical Biology

and Fundamental Medicine SB RAS, Novosibirsk, Russian Federation
MAITE MUNIESA Department of Microbiology, University of Barcelona, Barcelona, Spain
JENNIFER OTERO Departament de Genètica i Microbiologia, Universitat Autònoma de
Barcelona, Cerdanyola del Vallès, Spain
DIANA P. PIRES LIBRO – Laboratory of Research in Biofilms Rosário Oliveira, CEB –
JEAN-PAUL PIRNAY Laboratory for Molecular and Cellular Technology, Queen Astrid
CHRISTINE POURCEL Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS,
Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette Cedex, France
HILDE VAN RAEMDONCK Laboratory for Molecular and Cellular Technology, Queen Astrid
DOMINIC SAUVAGEAU Chemical and Materials Engineering, University of Alberta,
Edmonton, AB, Canada
SANNA SILLANKORVA CEB – Centre of Biological Engineering, University of Minho, Braga,
Portugal
IYO TAKEMURA-UCHIYAMA School of Veterinary Medicine, Azabu University, Sagamihara,
Kanagawa, Japan
NINA V. TIKUNOVA Laboratory of Molecular Microbiology, Institute of Chemical Biology and
Fundamental Medicine SB RAS, Novosibirsk, Russian Federation
IGOR TOLSTOY National Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, Bethesda, MD, USA
JUMPEI UCHIYAMA School of Veterinary Medicine, Azabu University, Sagamihara,
Kanagawa, Japan
TAKAKO UJIHARA Science Research Center, Kochi University, Kohasu, Oko-cho, Nankoku,
Kochi, Japan
GILBERT VERBEKEN Laboratory for Molecular and Cellular Technology, Queen Astrid
DANIEL DE VOS Laboratory for Molecular and Cellular Technology, Queen Astrid Military
Hospital, Brussels, Belgium
GORDON WHEAT PhageBiotics Research Foundation, Gray’s Harbor Community Hospital,
Aberdeen, WA, USA; Kaiser Permanente Northwest, Saint Peter Hospital Family Medicine
Residency, Olympia, WA, USA
Part I
Isolation of Bacteriophages
Chapter 1
Isolation of Bacteriophages for Fastidious Bacteria

Shigenobu Matsuzaki, Jumpei Uchiyama, Iyo Takemura-Uchiyama,
Takako Ujihara, and Masanori Daibata
Abstract
One of the most important factors for successful bacteriophage therapy is, undoubtedly, the isolation of
excellent therapeutic candidate bacteriophages. There are only a few reports about active bacteriophages in
the fastidious bacteria Helicobacter pylori. In this chapter, we describe a method for isolating and purifying
KHP30-like bacteriophages in H. pylori, which have lytic and pseudolysogenic life cycles.
Key words Helicobacter pylori, KHP30-like bacteriophage, Lytic and pseudolysogenic life cycles,
Isolation, Purification
1 Introduction
Helicobacter pylori is a Gram-negative, spiral, and microaerophilic

bacterium. It is well known as a causative agent in chronic and acute
gastritis, gastric ulcers, and gastric cancer [1, 2]. If the bacterium is
detected in the stomach by bacterial examination, its eradication
will be recommended using antimicrobial agents such as clarithro-
mycin, metronidazole, and amoxicillin. However, as antimicrobial
resistance (AMR) is also progressing in the case of H. pylori like
other bacterial cases, its eradication will become more difficult in
the future [2]. Therefore, the isolation of therapeutic candidate
bacteriophages for the eradication of H. pylori from the human
stomach is thought to be meaningful.
Despite the scarcity of reports about active H. pylori bacterio-
phages [3–9], KHP30 is one of the best characterized of these
bacteriophages [7, 8]. Bacteriophages that are genetically related
to KHP30 are called KHP30-like bacteriophages in this article.
Both KHP30 and KHP40, another KHP30-like bacteriophage,
have lytic and pseudolysogenic life cycles [7, 8, 10]. Although
lytic bacteriophages are thought to be suitable as therapeutic bac-
teriophages, mutant bacteriophages derived from them that have
Joana Azeredo and Sanna Sillankorva (eds.), Bacteriophage Therapy: From Lab to Clinical Practice, Methods in Molecular Biology,
vol. 1693, DOI 10.1007/978-1-4939-7395-8_1, © Springer Science+Business Media LLC 2018
3
4 Shigenobu Matsuzaki et al.
lost pseudolysogenic ability via spontaneous mutations may also be

useful for bacteriophage therapy.
In this chapter, we describe isolation and purification methods
of KHP30-like bacteriophages in H. pylori.
2 Materials
2.1 Bacteriophage 1. H. pylori strains.

Isolation 2. Brucella broth.
3. Equine serum (inactivated by heating for 30 min at 56 C).
4. Vancomycin (filtrated using a filter with a pore size of 0.2 μm,
10 mg/mL of water).
5. BEV: Brucella broth including 10% (vol/vol) equine serum
and 10 μg/mL of vancomycin (see Note 1).
6. BEV plate: BEV including 1.5% (wt/vol) agar (ca. 20 mL in a
plastic Petri dish with a diameter of 9–10 cm).
7. SBA (soft Brucella agar): Brucella broth including 0.5%
(wt/vol) agar and kept at 55 C in a long glass screw vial
(diameter, 1.8 cm; length, 15–18 cm) on a dry hot bath.
8. Sterile plastic tube (10, 50 mL) for culture.
9. Sterile syringe filter with a pore size of 0.45 μm and sterile
plastic syringe.
10. Sterile Conradi stick.
11. Micropipette (10 μL and sterile plastic tip for it).
12. Sterile plastic loop (10 μL).
13. Sterile plastic needle.
14. Dry hot bath: equipment for standing SBA-containing grass
vial at 55 C.
15. CO2 incubator.
16. Shaker in CO2 incubator.
17. Cooling centrifuge.
2.2 Purification 1. BCV: Brucella broth including 0.5% (wt/vol) β-cyclodextrin

of Bacteriophage and 10 μg/mL of vancomycin (see Note 2).
Particles 2. Polyethylene glycol 6000 and Tween 20.
3. TM buffer (10 mM Tris–HCl, 5 mM MgCl2, pH 7.2).
4. DNase I: Deoxyribonuclease I (10 mg/mL).
5. RNaseA: Ribonuclease A (10 mg/mL).
6. AAS solution (100 mM ammonium acetate, 10 mM NaCl,
1 mM MgCl2, 1 mM CaCl2, pH 7.2).
Preparation of H. pylori Bacteriophage 5
7. CsCl (ρ ¼ 1.7, 1.5, and ρ ¼ 1.3) in AAS solution.

8. Plastic syringe with a needle or dropper.
9. Dialyzing tube (cut-off molecular weight, 10,000).
10. Cooling centrifuge.
11. Ultracentrifuge.
12. Ultracentrifugation tube (6 mL).
3 Methods
This protocol shows a detailed description of the method previ-

ously described [8]. In this protocol, H. pylori strains are cultured
in air containing 10% CO2 at 37 C.
3.1 Preparation 1. H. pylori strains are shaken vigorously in 2 or 5 mL BEV in

of the Supernatant 10 or 50 mL plastic tubes for 2 days (see Notes 3 and 4).
of an H. pylori Culture 2. The strains are centrifuged at 7000 g (or lower) for 5 min at
4 C, to remove the residual host cells.
3. The supernatants are filtrated using a filter with a pore size of
0.45 μm with a plastic syringe (see Note 5).
3.2 Easy Screening 1. 0.5 mL of the host culture fully grown in BEV is placed on the
of Active BEV plate, and 5 mL of SBA is poured on it, followed by
Bacteriophages: Spot solidification (i.e., bilayer method) (see Note 6).
Test and Streak Test 2. For the spot test, 3 μL of the filtrate from Subheading 3.1, step
3 is spotted onto the surface of the upper layer, which includes
the host cells, using a micropipette (Fig. 1a). For the streak
test, several lines are gently streaked on the surface of the upper
layer using a plastic loop carrying 10 μL of the filtrate (Fig. 1b)
(see Note 7). Subsequently, the plate is incubated for 3 days.
3. Results of the spot test: the appearance of a spot shows that the
filtrate possibly includes bacteriophages. However, it does not
necessarily indicate that the bacteriophages have propagated in
the host. Therefore, after the spot test, plaque-forming activity
needs to be examined, to detect active bacteriophages.
4. Results of the streak test: the observation of lysis lines to the
end, or lysis lines and plaques following them, implies that the
filtrate includes active bacteriophage(s) (Fig. 1b). Conversely,
the presence of lysis lines without plaques following them may
indicate lysis from without, in which bacteriophages adsorb to
the host but do not propagate in it. The absence of lysis lines
indicates that there may not be any bacteriophage infection in
the host.
Fig. 1 Spot test and streak test using the supernatants of cultures of the H. pylori strains NY43 and KMT83, in
which KHP30-like bacteriophages, KHP30 and KHP40, are released, respectively. Upper panel: spot test
(3 μL). Lower panel: streak test (10 μL). Left panel: schematic representation of the procedures. Right panel:
results obtained after 3 days
3.3 Isolation 1. Filtrates in which the presence of bacteriophages has been

of H. pylori shown are serially diluted in 0.5 mL BEV (e.g., 1, 101,
Bacteriophages by 102, 103, 104).
Single-Plaque 2. 0.1 mL of the diluted samples and 0.5 mL of the host culture
Isolation Procedure fully grown in BEV, which showed sensitivity to the active
bacteriophage, are placed on the BEV plate, and 5 mL SBA is
then poured onto them and mixed evenly. After solidification,
the plates are incubated for 3 days.
3. A well-separated single plaque is picked up using a needle,
suspended in 0.5 mL of BEV medium, and diluted to 101,
102, 103, and 104 in BEV (see Note 8).
4. 0.1 mL of each bacteriophage dilute and 0.5 mL of the host
culture fully grown in BEV are placed on the BEV plate, and
SBA is poured onto it. After solidification, the plates are incu-
bated again for 3 days.
5. Another single plaque is picked up using a needle, diluted, and
cultured with the host as described above. The single plaque
isolation procedure is repeated at least three times, to avoid
contamination by other bacteriophages.
3.4 Preparation of an 1. A plaque that appears in the third cycle after the procedure
H. pylori described above is picked up using a needle and transferred into
Bacteriophage Stock 0.5–1 mL of BEV medium.
by Confluent Lysis 2. 0.1 mL of the bacteriophage suspension and 0.5 mL of the host
Procedure culture are placed on five BEV plates, and SBA is poured onto
each plate and mixed. After solidification, the plates are incu-
bated for 3 days.
3. 10 mL of liquid BEV are poured onto one of the plates in which
the host has been lysed completely. After the upper layer is
crushed with a sterile Conradi stick, it is transferred onto the
next plate. This procedure is repeated. Finally, all of the crushed
upper layers with the medium are transferred into a 50 mL
plastic tube and are vortexed vigorously.
4. The tube is centrifuged at 7000 g for 5 min at 4 C, and the
supernatant is filtrated using a filter with a pore size of 0.45 μm.
5. The bacteriophages are probably stable at 4 C for at least
2 years. For long-term storage, 0.5 mL of the filtrate is mixed
with 0.5 mL of 60% (vol/vol) glycerol and is placed at 80 C.
3.5 Preparation 1. 5–10 mL of H. pylori culture that has been grown fully in BEV
of Purified and 0.1–0.2 mL of the bacteriophage stock (usually
Bacteriophage ca. 1–5 109 pfu/mL) are transferred to 250–500 mL of
Particles Using CsCl BCV medium in a flask and the culture is vigorously shaken
Density Gradient for 2–3 days (see Note 9).
Ultracentrifugation 2. The culture is centrifuged at 10,000 g for 10 min at 4 C, to
precipitate the host cell debris.
3. 10% (wt/vol) polyethylene glycol 6000, 3 M NaCl, and 1%
(vol/vol) Tween 20 are added to the supernatant and are
solubilized well (see Note 10).
4. The mix is centrifuged at 10,000 g for 20–40 min at 4 C.
5. The precipitate is resuspended in 2–3 mL of TM buffer includ-
ing DNaseI (final concentration, 100 μg/mL) and RNaseI
(final concentration, 100 μg/mL), followed by incubation at
37 C for 60 min.
6. 1 mL of the sample is overlaid on top of a stepwise CsCl density
gradient consisting of 0.5 mL (ρ ¼ 1.7), 1.5 mL (ρ ¼ 1.5),
and 2.5 mL (ρ ¼ 1.3) from the bottom upwards (Fig. 2b)
(see Note 11).
7. The tube is centrifuged at 100,000 g for 60 min at 4 C.
8. The purified bacteriophage (white band) is obtained using a
syringe and needle or a plastic dropper.
9. The purified bacteriophage band is transferred to a dialyzing
tube (cut-off molecular weight, 10,000) and is dialyzed against
1000 mL of AAS at 4 C for 60 min.
Fig. 2. KHP30 bacteriophage band in two types of stepwise CsCl density gradient ultracentrifugation. (a) Usual
method: 2.5 mL (ρ ¼ 1.7), 2.5 mL (ρ ¼ 1.5), and 2.5 mL (ρ ¼ 1.3) from the bottom. (b) Modified method for
the preparation of KHP30-like bacteriophages: 0.5 mL (ρ ¼ 1.7), 1.5 mL (ρ ¼ 1.5), and 2.5 mL (ρ ¼ 1.3) from
the bottom. Left and right in a and b depict the experiment before and after ultracentrifugation, respectively.
Black arrow, bacteriophage band. White arrow, debris. (c) Electron micrograph of KHP30 purified using the
unusual method
10. The purified bacteriophage sample can be used for electronic

microscope observation, DNA preparation, or virion protein
preparation (e.g., Fig. 2c).
4 Notes
1. In the preparation of 1000 mL, 900 mL of water including

28 g of Brucella broth powder (and 15 g agar) are autoclaved
and, after cooling to a temperature that allows hand touching,
100 mL of equine serum and vancomycin (final concentration,
10 μg/mL) are added.
2. 1000 mL of water including 28 g of Brucella broth powder and
5 g of β-cyclodextrin are autoclaved and, after cooling, vanco-
mycin (final concentration, 10 μg/mL) is added.
3. Aeration is very important for H. pylori culture. A 10 or 50 mL
plastic tube is used for 2 or 5 mL culture, respectively, and a
shaking speed >200 rpm is recommended.
4. As active KHP30-like H. pylori bacteriophages are found rarely
(about 1%), the examination of as many as possible H. pylori
strains is recommended. KHP30-like bacteriophages do not
seem to be induced by mitomycin C addition.
5. Usage of a filter with a pore size of 0.2 μm is not recommended
because large bacteriophages (such as T4-related bacterio-
phages) cannot pass through the filter perfectly. Therefore, in
general, the use of a 0.45 μm filter is recommended for the
examination of unknown bacteriophages.
6. H. pylori strains seem commonly to have strong restriction-

modification systems. Therefore, as many as possible host
strains should be examined during the screening for KHP30-
like bacteriophages.
7. The spot test is suitable to examine many filtrates, but the
plaque-forming activity needs to be checked. Conversely, in
the streak test, plaque-forming activity can be checked in
one step.
8. If several types of plaques showing a different morphology or
diameter appear, all types of plaques should be picked up
independently because they may be taxonomically different
from each other.
9. BCV medium is more suitable than BEV medium for large-
scale bacteriophage preparation because the presence of equine
serum in the medium seems to produce a lot of debris.
10. The addition of 3 M NaCl (usually 0.5 M) and 1% Tween
20 promotes the separation of the bacteriophages from the
debris.
11. This is a modified stepwise CsCl density gradient ultracentrifu-
gation. As the density of KHP30-like bacteriophages is low, the
bacteriophage band is hardly separated from the upper debris
layer in the usual method, which uses an equal volume of CsCl
solution with ρ ¼ 1.7, 1.5, and 1.3 from the bottom (Fig. 2a
and b).
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research

(C) (26461504), Japan Society for the Promotion of Science and
the Institute for Fermentation, Osaka, Japan.
References
1. Atherton JC, Blaser MJ (2009) Coadaptation 4. Heintschel von Heinegg E, Nalik HP, Schmid
of Helicobacter pylori and humans: ancient his- EN (1993) Characterisation of a Helicobacter
tory, modern implications. J Clin Invest pylori phage (HP1). J Med Microbiol
119:2475–2487 38:245–249
2. Smith SM, O’Morain C, McNamara D (2014) 5. Wan XQ, Tang DS, Liu AP, Tan SY, Li WK,
Antimicrobial susceptibility testing for Helico- Kuang J, Li HM (2011) Isolation of a wild-
bacter pylori in times of increasing antibiotic type virulent phage of Helicobacter pylori and
resistance. World J Gastroenterol its simulated treatments of gastrointestinal Hp
20:9912–9921 in vitro (Chinese). Nan Fang Yi Ke Da Xue Xue
3. Schmid EN, von Recklinghausen G, Ansorg R Bao 31:304–307
(1990) Bacteriophages in Helicobacter (Cam- 6. Luo CH, Chiou PY, Yang CY, Lin NT (2012)
pylobacter) pylori. J Med Microbiol Genome, integration, and transduction of a
32:101–104 novel temperate phage of Helicobacter pylori. J
Virol 86:8781–8792
7. Uchiyama J, Takeuchi H, Kato S, Takemura- Helicobacter pylori isolated from Egypt. Fut
Uchiyama I, Ujihara T, Daibata M, Matsuzaki Virol 8:821–826
S (2012) Complete genome sequences of two 10. Uchiyama J, Takemura-Uchiyama I, Kato S,
Helicobacter pylori bacteriophages isolated Takeuchi H, Sakaguchi Y, Ujihara T,
from Japanese patients. J Virol Daibata M, Shimakura H, Okamoto N,
86:11400–11401 Sakaguchi M, Matsuzaki S (2016) Screening
8. Uchiyama J, Takeuchi H, Kato S, Gamoh K, of KHP30-like prophages among Japanese
Takemura-Uchiyama I, Ujihara T, Daibata M, Helicobacter pylori strains, and genetic analysis
Matsuzaki S (2013) Characterization of Heli- of a defective KHP30-like prophage sequence
cobacter pylori bacteriophage KHP30. Appl integrated in the genome of the H. pylori strain
Environ Microbiol 79:3176–3184 NY40. FEMS Microbiol Lett 363(16):pii:
9. Abdel-Haliem MEF, Askora A (2013) Isola- fnw157
tion and characterization of bacteriophages of
Chapter 2
Isolation of Bacteriophages of the Anaerobic Bacteria

Bacteroides
Cristina Garcı́a-Aljaro, Maite Muniesa, and Juan Jofre
Abstract
Here we describe the detection, enumeration, and isolation of bacteriophages infecting Bacteroides. The
method is based on the infection of Bacteroides host strains and the production of visible plaques in a
confluent lawn of the host strain using the double-layer agar method. This is a straightforward methodol-
ogy that can be applied for the detection, enumeration and isolation of bacteriophages for other anaerobic
bacteria, using an appropriate host strain and culture conditions. In the case of bacteriophages of Bacter-
oides the results can be obtained in less than 24 h, although the time could vary depending on the growth
rate of the host strain.
Key words Bacteriophage, Double-layer agar method, Anaerobic, Lytic plaques, Bacteroides
1 Introduction
A number of bacteriophages have been incorporated in several

regulations as indicators of microbial water quality to validate and
verify water treatment processes [1–6]. In this respect, the presence
of bacteriophages infecting Bacteroides, one of the most abundant
species in the gut, in an environmental sample indicates the pres-
ence of fecal pollution of human or animal origin. Different studies
have pointed out that bacteriophages infecting different Bacteroides
species can be used for microbial source tracking purposes [7–14]
although certain geographic variation has also been reported [8, 9,
13, 14]. Despite different molecular detection methods have been
reported, detection of bacteriophages using the double-layer agar
method with an appropriate host strain is a straightforward and
nonexpensive methodology which, besides detection of infecting
bacteriophages and evaluation of the microbiological water quality,
allows their isolation for different purposes. For example, bacter-
iophages of Bacteroides can be isolated and used to study the
bacteriophage receptors, which are different cell surface compo-
nents that may be involved in the virulence of different Bacteroides.
Joana Azeredo and Sanna Sillankorva (eds.), Bacteriophage Therapy: From Lab to Clinical Practice, Methods in Molecular Biology,
vol. 1693, DOI 10.1007/978-1-4939-7395-8_2, © Springer Science+Business Media LLC 2018
11
12 Cristina Garcı́a-Aljaro et al.
Fig. 1 Typical lytic plaques of phages infecting Bacteroides fragilis grown on

semisolid BPRMA
In this chapter, we report a methodology for the detection and

enumeration of bacteriophages infecting Bacteroides based on the
double-layer agar method, as an example of bacteriophages for
anaerobic bacteria. The most abundant bacteriophages infecting
Bacteroides belong to the family Siphoviridae with flexible tail
(dsDNA, long noncontractile tails, capsids up to 60 nm) (Fig. 1).
The method relies on the infection of selected bacterial host and the
production of visible plaques (clearance zones) in a confluent host
bacteria lawn grown under appropriate culture conditions. Bacter-
iophages detected are those virulent infecting Bacteroides by attach-
ing to molecules present in the bacteria cell wall. Virulent
bacteriophages may lyse the host cell in 30–40 min under optimal
conditions. They produce clear plaques which do not differ very
much in size and morphology (Fig. 2). This method can be applied
to all types of water, sediments and sludge extracts, as well as
shellfish extracts and has been standardized by the International
Standard Organization [15].
2 Materials
2.1 Handling 1. Bacteroides fragilis RYC2056 [10], although other Bacteroides

and Culturing the Host species or strains can be used as long as they can grow using the
Strain same medium and culture conditions. For example, B. fragilis
HSP40 (ATCC 51477) [16], B. thetaiotaomicron GA17 and
others have been successfully used following the same method
with minor modifications [17].
2. Basal Bacteroides Bacteriophage Recovery Medium Broth
(BPRMB) [18]: weight 10 g meat peptone, 10 g casein pep-
tone, 2 g yeast extract, 5 g NaCl, 0.5 g monohydrated
Bacteriophages of Bacteroides 13
Fig. 2 Electron micrographs of phages infecting Bacteroides fragilis and Bacteroides thetaiotaomicron.
Pictures show phages alone or attached to particles. Negative staining with 2% (wt/vol) KOH phosphotungstic
acid (pH 7.2). Bar 200 nm
L-cystein, 1.8 g glucose, 0.12 g MgSO4·7H2O, and add

1000 mL of distilled water. Dissolve the ingredients and add
1 mL of CaCl2 stock solution containing 0.05 g/mL prepared
as described below. Sterilize in the autoclave at 121 C for
15 min. Store in the dark at 4 C for no longer than 1 month
(see Note 1).
The complete broth (Table 1) should be prepared immediately
before use by adding aseptically 10 mL of hemin solution and
25 mL of disodium carbonate solution to 1000 mL of basal
broth (see Note 2). The pH should be adjusted to 6.8 by
adding HCl (e.g. 2.5 mL of HCl 35% (vol/vol)).
3. CaCl2 stock solution: weight 5 g of CaCl2·2H2O in 100 mL of
distilled water while heating gently. Cool to room temperature
and filter sterilize through a 0.2 μm pore size membrane filter
(high-binding protein cellulose ester membrane) [19]. Store in
the dark at 4 C for no longer than 6 months (see Note 1).
4. Hemin solution: weight 0.1 g of hemin, add 0.5 mL of NaOH
1 M and 99.5 mL of distilled water. Dissolve by magnetic
stirring (see Note 3). The solution can be filter-sterilized
Table 1
Composition of the Bacteroides phage recovery medium broth.
Meat peptone 10 g
Casein peptone 10 g
Yeast extract 2g
NaCl 5g
Monohydrated L-cystein 0.5 g
Glucose 1.8 g
MgSO4.7H2O 0.12 g
CaCl2 solution (0,05 g/mL) 1 mL
a
Hemin solution 10 mL
a
Na2CO3 solution 25 mL
Distilled water 1000 mL
a
Add aseptically immediately before use.
through a 0.2 μm pore size high protein binding membrane

filter, or sterilized in the autoclave at 121 C for 15 min. Store
at room temperature for no longer than 6 months.
5. Disodium carbonate solution (1 M) (Na2CO3): weight 10.6 g
of Na2CO3 in 100 mL of distilled water and filter-sterilize
through a 0.2 μm pore size high protein binding membrane
filter. Store at room temperature for no longer than 6 months.
6. Nalidixic acid solution: weight 250 mg Nalidixic acid in 2 mL
of NaOH (1 M), then add 8 mL of distilled water until
completely dissolved. Filter-sterilize through a 0.2 μm pore
size high protein binding membrane filter. Store at 4 C for
no longer than 8 h or at 20 C for no longer than 6 months.
7. Kanamycin monosulfate: weight 1.25 g of kanamycin mono-
sulfate in 10 mL of distilled water and filter-sterilize through a
0.2 μm pore size high protein binding membrane filter (see
Note 4). Store at 4 C for no longer than 8 h or at 20 C
for no longer than 6 months.
8. Bacteroides Bacteriophage Recovery Medium Agar (BPRMA):
Add 12–20 g of agar (depending on gel strength of agar) to
1000 mL of basal broth (not autoclaved). Sterilize by autoclaving
at 121 C for 15 min. Cool down to between 45 and 50 C and
add aseptically 10 mL of hemin solution and 25 mL of Na2CO3
solution to prepare the complete BPRMA (see Note 2). The pH
should be adjusted to 6.8 by adding HCl (e.g. 2.5 mL of HCl 35%
(vol/vol)). Pour into Petri dishes (20 mL in dishes of 9 cm

diameter). Allow to solidify and store in the dark at 4 C for no
longer than two months (see Note 1). Place the plates at room
temperature 1–2 h before use to dry them.
2.2 Host Strain Stock 1. Bacteroides host strain.

Culture Preparation 2. BPRMB tubes (see Subheading 2.1).
3. BPRMA plates (see Subheading 2.1).
4. Cryoprotector: 10% (wt/vol) bovine serum albumin, 20%
(wt/vol) sucrose in water. Sterilize using high protein binding
0.2 μm pore-size cellulose ester membrane filters.
5. Sterile swabs.
6. Anaerobic jar or chamber.
7. Sterile screw-capped vials.
8. Sterile tips.
9. Incubator at 37 C.
10. 70 C freezer.
2.3 Preparation 1. Bacteroides host strain stock culture.

of Host Strain 2. BPRMB tubes (see Subheading 2.1).
Inoculum Culture
for Bacteriophage
Analysis 4. Sterile screw-capped vials.
5. Sterile tips.
8. Spectrophotometer.
2.4 Bacteriophage 1. Bacteroides host strain inoculum culture.

Presence/Absence 2. BPRMA plates (see Subheading 2.1).
Test
3. Semisolid Bacteroides Bacteriophage Recovery Medium Agar
(ssBPRMA) tubes: Basal agar medium, agar (6–10 g), depend-
ing on the gel strength (see Note 5). Sterilize by autoclaving at
121 C for 15 min and distribute in volumes of 50 mL. Allow
to solidify and store at 4 C for no longer than two months.
Immediately before use, melt the basal medium, allow to cool
down to 45–50 C and add aseptically 10 mL of hemin solution
and 25 mL of Na2CO3 solution to prepare the complete
ssBPRMA (see Note 2). The pH should be adjusted to 6.8 by
adding HCl (e.g. 2.5 mL of HCl 35% (vol/vol)).
4. Bacteriophage suspension/samples.
5. Sterile tips.

8. Incubator at 45–50 C.
2.5 Bacteriophage 1. Bacteroides host strain inoculum culture.

Enumeration 2. BPRMA plates (see Subheading 2.1).
3. ssBPRMA tubes (see Subheading 2.4).
4. Sterile tips.
2.6 Isolation 1. Bacteriophage plaques.

of Bacteriophages 2. SM buffer: weight 5.8 g of NaCl, 2 g of MgSO4∙7H2O, 0.1 g
from Plaques of gelatin, and add 50 mL of Tris–HCl 1 M at pH 7.5. Adjust
to 1000 mL with double distilled water. Sterilize in autoclave at
121 C for 15 min.
3. BPRMB tubes (see Subheading 2.1).
5. ssBPRMA tubes (see Subheading 2.4).
6. Chloroform.
7. Sterile tubes.
8. Sterile needle.
9. Sterile tips.
10. 0.2 μm pore-size low protein binding membrane filter (PVDF
or PES).
12. Bench centrifuge.
2.7 Preparation of a 1. Bacteriophage suspension.

High-Titer 2. Bacteroides host strain inoculum culture.
Bacteriophage
Suspension in Solid
Agar Media 4. ssBPRMA tubes (see Subheading 2.4).
5. SM buffer.
6. Chloroform.
7. Sterile tubes.
8. Sterile needle.
9. Sterile pipette.
10. Sterile tips.
11. 0.2 μm pore-size low protein binding membrane filter (PVDF
or PES).

14. Bench centrifuge.
2.8 Preparation 1. Reference bacteriophage: Bacteriophage B56-3 (ATCC

of Reference 700786-B1) can be used as reference bacteriophage for
Bacteriophage Stock B. fragilis RYC2056, and bacteriophage B40-8 (ATCC
Culture 51477-B1) for B. fragilis HSP40 (ATCC 51477). Reference
bacteriophages have also been isolated for other hosts but to
the best of our knowledge they have not been deposited yet in
culture collections.
2. Peptone saline: weight 1 g of peptone and 8.5 g of NaCl, and
add 1000 mL of distilled water, adjust pH at 7.2. Sterilize in
autoclave at 121 C for 15 min.
3. Sterile glycerol.
4. Sterile tubes.
5. Sterile tips.
6. 70 C freezer.
3 Methods
3.1 Handling Bacteroides do not require very strict anaerobic conditions for
and Culturing the Host handling or for growing: the bacterium can be handled shortly in
Strain the presence of air but agar plates (BPRMA) should be incubated in
anaerobic cabinets, jars, or bags containing anaerobiosis generators
and anaerobic indicators. Liquid cultures in BPRMB can be per-
formed in glass tubes, flaks or bottles by filling them completely
with medium and closing them with screwing caps, without the
need of incubating them under anaerobic conditions. Of course,
they can be handled and incubated under anaerobic conditions.
Other anaerobic bacteria may require more strict anaerobic condi-
tions and different growth media and culture conditions.
3.2 Host Strain Stock 1. Inoculate a BPRMA plate with the corresponding host strain
Culture Preparation (cover all the plate twice with a sterile swab to ensure the
maximal growth) and incubate 24–48 h at 37 C in anaerobic
jar or chamber.
2. Inoculate 10 mL of BPRMB with at least 1/4 of the cells
previously grown the BPRMA plate and incubate overnight at
37 C (see Notes 6 and 7).
3. Mix culture and cryoprotector in a 1:1 ratio (vol/vol), avoiding
bubble formation. Distribute into sterile screw-capped vials in
aliquots of ca. 0.5 mL and store at 70 C.
3.3 Preparation 1. Thaw one vial of stock culture at room temperature and streak
of Host Strain on a plate of BPRMA covering all the plate. Incubate 24–48 h
Inoculum Culture at 37 C. After 24 h of incubation check if a dense bacterial
for Bacteriophage growth is visible. Otherwise, incubate for extra 24 h at 37 C.
Analysis The plate should be completely covered of cell material before
inoculating the strain in step 2.
2. Inoculate at least 1/4 of the cell material grown on the plate in
10 mL of prewarmed BPRMB and incubate overnight at 37 C
(see Note 8).
3. Transfer this culture to fresh prewarmed BPRMB screw capped
tube in a ratio 1:10 (vol/vol). Fill completely the tube with
BPRMB and incubate at 37 C. After 2 h measure absorbance
every 30 min until reaching approximately 5 108 cfu/mL
(OD620 0.3–0.5 based on data obtained in our laboratory) (see
Note 9).
4. Take the inoculum culture from the incubator and quickly cool
the culture by placing it in ice. Use within 6 h.
3.4 Bacteriophage 1. Add 1 mL of the inoculum culture to 2.5 mL of melted

Presence/ ssBPRMA, previously cooled to 45–50 C (see Note 10).
Absence Test 2. Mix well and pour onto a BPRMA plate.
3. Allow to solidify and add a drop (up to 20 μL) of the bacterio-
phage suspension or filtered samples to be tested (see Note 11).
Allow the drop to adsorb on the agar for at least 15 min.
4. Incubate the plates upside down overnight at 37 C in anaero-
bic conditions (see Subheading 3.1).
5. Detection of a clearance zone (plaque formation) (see Note 12).
3.5 Bacteriophage 1. Add 1 mL of the inoculum culture grown at OD620 0.3–0.5,

Enumeration and 1 mL of the sample to be tested to 2.5 mL of melted
ssBPRMA (see Notes 10 and 13).
2. Mix well avoiding bubble formation and pour onto a BPRMA
plate.
3. Allow to solidify and incubate plates upside down overnight at
37 C in anaerobic conditions (see Subheading 3.1).
4. Count the number of lytic plaques and calculate the number of
plaque forming units (pfu) per mL depending on the sample
dilution analyzed.
3.6 Isolation 1. Carefully excise the desired plaque from the over layer of
of Bacteriophages semisolid agar using a sterile needle.
from Plaques 2. Resuspend the bacteriophages in the plaque in SM buffer.
3. Add chloroform at 1:10 (vol:vol). Mix vigorously for 5 min.
Centrifuge at 16,000 g for 5 min.
4. Recover supernatant.
5. Inoculate 1 mL of supernatant with 9 mL of host strain inocu-
lum, fill the tube with BPRMB to the top, and incubate over-
night at 37 C.
Centrifuge at 16,000 g for 5 min. Carefully transfer the
aqueous supernatant to a sterile empty agar plate avoiding
solvent phase.
7. Filter the supernatant through a 0.2 μm pore-size low protein
binding membrane filter (PVDF or PES membranes) [19] (see
Note 14).
8. Enumerate the bacteriophages in the suspension. If higher
bacteriophage titer is desired, repeat steps 5–8 using 1 mL of
the suspension.
3.7 Preparation of a 1. Use 1 mL of bacteriophage suspension obtained in Subheading

High-Titer 3.6 (step 4) and proceed as for bacteriophage enumeration.
Bacteriophage Prepare one control plate without bacteriophage suspension
Suspension in Solid (containing only the host strain and ssBPRMA.
Agar Media 2. After incubation, confluent lysis should be expected in compar-
ison with the bacterial control plate. Add 5 mL of SM buffer
and drop it onto the surface of the plate with confluent lysis.
Incubate 15 min at 4 C.
3. Carefully recover the liquid from the surface with a sterile
pipette.
Centrifuge at 16,000 g for 5 min.
5. Recover supernatant and filter it through 0.2 μm diameter
pore-size filter.
3.8 Preparation 1. Dilute bacteriophage suspension in peptone saline solution to

of Reference contain between 40 and 100 pfu/mL.
Bacteriophage Stock 2. Add sterile glycerol to the bacteriophage suspension (final
Culture (See Note 13) glycerol concentration should be 10% (vol/vol)).
3. Distribute into aliquots of 1 mL and store at 70 C.
4. Check the intra- and inter-vial homogeneity (see Note 15).
4 Notes
1. For long-term storage, ISO method recommends storage at

4 C in the dark to prevent contamination or undesirable
changes in the solution.
2. Bacteroides growth is slower as other possible contaminants.

Therefore to avoid competition with other microorganisms
present in the samples the use of antibiotics is recommended.
One of the most common contaminants are Gram-positive
cocci. To prevent contamination it is recommended to add
Kanamycin monosulfate (final concentration of 100 μg/mL)
and Nalidixic acid (final concentration of 100 μg/mL) to the
complete broth. Since not all strains of Bacteroides display the
same resistance range to both antibiotics, how these can affect
the growth of the strain should be tested previously.
3. Complete dissolution of hemin is necessary and may last
between 30 and 60 min.
4. Many suppliers of kanamycin sulfate contain less than 100%
active Kanamycin. Make the necessary corrections to reach
1.25 g of active kanamycin monosulfate to 10 mL of water.
5. The gel strength of ssBPRMA is critical to obtain good results
and if possible different concentrations should be tested.
Choose the agar concentration that produces the highest
plaque counts but also control plaque-size to either reduce
confluence or very small plaques. The most commonly used
in our lab is 7 g/L.
6. Depending on the growth of the host strain on BPRMA,
inoculate 1/8 or more of the cell material grown on the surface
of BPRMA, using a sterile cotton swab (e.g. in case of dense
growth use 1/8 to inoculate 10 mL BPRMB, in case of poor
growth use 1/2 of the full slant).
7. To ensure anaerobic conditions, completely fill the tube with
BPRM broth and close the screw-capped tube. Otherwise a
tube not completely filled and not completed sealed could be
incubated using an anaerobic jar.
8. The overnight liquid culture can be prepared directly from the
stock (without intermedium stage in an agar plate) in a ratio
1:10 (vol:vol), into to a screw-capped tube.
9. We strongly recommend performing a growth curve of the
host strain in order to calculate the correspondence between
CFU/mL and OD620 nm, due to possible variations in spectro-
photometers. If the cell density of approximately
5 108 CFU/mL is not reached within three hours, it is
possible to increase the amount of working culture transferred
into the BPRMB to a ratio 1.5:10 (vol:vol) or the
incubation time.
10. Immediately before use, hemin and Na2CO3 solutions, as well
as antibiotics (if desired) should be added to ssBPRM. The
accuracy of counting bacteriophages depends on the stability of
the host strain. In order to check the stability of the strain a
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Matilda threw as many obstacles as she could in the way of the
completion of the nuptial ceremony. At last this solemn matter was
definitively settled to come off at Rouen, on the 26th of August, 1127.
Geoffrey must have been a knight before his marriage with Matilda.
However this may be, he is said to have been created an English
knight in honor of the occasion. To show how he esteemed the
double dignity of knight and husband, he prepared himself for both,
by first taking a bath, and afterward putting on a clean linen shirt.
Chroniclers assure us that this is the first instance, since the
Normans came into England, in which bathing is mentioned in
connection with knighthood. Over his linen shirt Geoffrey wore a
gold-embroidered garment, and above all a purple mantle. We are
told too that he wore silk stockings, an article which is supposed to
have been unknown in England until a much later period. His feet
were thrust into a gay pair of slippers, on the outside of each of
which was worked a golden lion. In this guise he was wedded to
Matilda, and never had household lord a greater virago for a lady.
From this circumstance the Knights of the Bath are said to have had
their origin. For a considerable period, this order of chivalry ranked
as the highest military order in Europe. All the members were
companions. There was but one chief, and no knight ranked higher,
nor lower, than any other brother of the society. The order,
nevertheless, gradually became obsolete. Vacancies had not been
filled up; that Garter had superseded the Bath, and it was not till the
reign of George II. that the almost extinct fraternity was renewed.
Its revival took place for political reasons, and these are well detailed
by Horace Walpole, in his “Reminiscences of the Courts of George
the First and Second.” “It was the measure,” he says, “of Sir Robert
Walpole, and was an artful bunch of thirty-six ribands, to supply a
fund of favors, in lieu of places. He meant, too, to stave off the
demand for garters, and intended that the red should have been a
stage to the blue; and accordingly took one of the former himself. He
offered the new order to old Sarah, Duchess of Marlborough, for her
grandson the Duke, and for the Duke of Bedford, who had married
one of her granddaughters. She haughtily replied, that they should
take nothing but the Garter. ‘Madam,’ said Sir Robert, coolly, ‘they
who take the Bath will the sooner have the Garter.’ The next year he
took the latter himself, with the Duke of Richmond, both having been
previously installed knights of the revived institution.”
Sir Robert respected the forms and laws of the old institution, and
these continued to be observed down to the period following the
battle of Waterloo. Instead of their creating a new order for the
purpose of rewarding the claimants for distinction, it was resolved to
enlarge that of the Bath, which was, therefore, divided into three
classes.
First, there was the Grand Cross of the Bath (G. C. B.), the reward of
military and diplomatic services.
The second class, of Knights Commanders (C. B.), was open to
those meritorious persons who had the good luck to hold
commissions not below the rank of Lieutenant-Colonel or Post-
Captain. The members of this class rank above the ordinary knights-
bachelors.
The third class, of Knights Companions, was instituted for officers
holding inferior commissions to those named above, and whose
services in their country’s cause rendered them eligible for
admission.
These arrangements have been somewhat modified subsequently,
and not without reason. Henry VIIth’s Chapel in Westminster Abbey
is the locality in which the installation of the different knights takes
place. The statutes of the order authorize the degradation of a knight
“convicted of heresy against the Articles of the Christian religion;” or
who has been “attainted of high treason,” or of “cowardly flying from
some field of battle.” It is rather curious that felony is not made a
ground of degradation. The Duke of Ormond was the last Knight of
the Garter who was degraded, for treason against George I.
Addison, after the degradation, invariably speaks of him as “the late
Duke.” A more grievous offender than he was that Earl of Somerset,
who had been a reckless page, and who was an unworthy Knight of
the Garter, under James I. He was convicted of murder, but he was
not executed, and to the day of his death he continued to wear the
Garter, of which he had been pronounced unworthy. The last
instances of degradation from the Order of the Bath were those of
Lord Cochrane (in 1814), for an alleged misdemeanor, and Sir Eyre
Coote, two years subsequently. In these cases the popular judgment
did not sanction the harsh measures adopted by those in authority.[1]
[1] Subsequently, the Prince Regent ordered the name of Captain
Hanchett to be erased from the roll of the Bath, he having been
struck off the list of Captains in the Royal Navy.
In olden times, the new Knights of the Bath made as gallant display
in public as the Knights of the Garter. In reference to this matter, Mr.
Mackenzie Walcott, in his “Westminster,” cites a passage from an
author whom he does not name. The reverend gentleman says: “On
Sunday, July 24th, 1603, was performed the solemnity of Knights of
the Bath riding honorably from St. James’s to the Court, and made
show with their squires and pages about the Tilt-yard, and after went
into the park of St. James, and there lighted from their horses and
went up into the King’s Majesty’s presence, in the gallery, where they
received the order of Knighthood of the Bath.”
The present “Horse-Guards” occupies a portion of the old Tilt-yard;
but for the knightly doings there, and also in Smithfield, I must refer
all curious readers to Mr. Charles Knight’s “Pictorial History of
London.”
The Order of the Thistle, if Scottish antiquaries may be
credited, is almost as ancient as the times in which the first thistle
was nibbled at by the primitive wild-ass. Very little, however, is
known upon the subject, and that little is not worth repeating. The
earliest certain knowledge dates from Robert II., whose coins bore
the impress of St. Andrew and his cross. James III. is the first
monarch who is known to have worn the thistle, as his badge. There
is no evidence of these emblems being connected with knighthood
until the reign of James V. The Reformers, subsequently, suppressed
the chivalric order, as popish, and it was not till the reign of James II.
of England that the thistle and chivalry again bloomed together. The
order is accessible only to peers. A commoner may have conferred
more honor and service on his country than all the Scottish peers put
together, but no amount of merit could procure him admission into
the Order of the Thistle. Nevertheless three commoners did once
belong to it; but their peculiar merit was that they were heirs
presumptive to dukedoms.
Ireland was left without an order until the year 1783, when George
III. good-naturedly established that of St. Patrick, to the great delight
of many who desired to be knights, and to the infinite disgust of all
who were disappointed. Except in name and local circumstances
there is nothing that distinguishes it from other orders.
I must not conclude this section without remarking, that shortly after
the sovereignty of Malta and the Ionian Isles was ceded to Great
Britain, the Order of St. Michael was instituted in 1818, for the
Purpose of having what Walpole calls “a fund of ribands,” to reward
those native gentlemen who had deserved or desired favors, if not
places.
The Order of the Guelphs was founded by the Prince Regent in
1815. George III. had designed such an order for the most
distinguished of his Hanoverian subjects. Down to the period of the
accession of Queen Victoria, however, the order was conferred on a
greater number of Englishmen than of natives of Hanover. Since the
latter Kingdom has passed under the rule of the male heir of the line
of Brunswick, the order of Guelph has become a foreign order.
Licenses to accept this or any other foreign order does not authorize
the assumption of any style, appellation, rank, precedence, or
privilege appertaining unto a knight-bachelor of these realms. Such
is the law as laid down by a decision of Lord Ellenborough, and
which does not agree with the judgment of Coke.
The history of foreign orders would occupy too much of my space;
but there is something so amusing in the history of an order of
knights called “Knights of the Holy Ampoule,” that a few words on the
subject may not be unacceptable to such readers as are
unacquainted with the ephemeral cavaliers in question.
THE KNIGHTS OF THE “SAINTE AMPOULE.”
“Mais ce sont des chevaliers pour rire.”— Le Sage.
There have been knights who, like “special constables,” have

been created merely “for the nonce;” and who have been as
ephemeral as the shortlived flies so called. This was especially the
case with the Knights of the Holy “Ampoule,” or anointing oil, used at
the coronation of the kings of France.
This oil was said to have been brought to St. Remy (Remigius) by a
dove, from Heaven, and to have been placed by the great converter
of Clovis, in his own tomb, where it was found, by a miraculous
process. St. Remy himself never alluded either to the oil or the story
connected with it. Four centuries after the saint’s death the matter
was first spoken—nay, the oil was boldly distilled, by Hinckmar,
Archbishop of Rheims. This archi-episcopal biographer of St. Remy
has inserted wonders in the saint’s life, which staggered, while they
amused, the readers who were able to peruse his work by fireside, in
castle-hall, or convert refectory. I can only allude to one of these
wonders—namely, the “Sainte Ampoule.” Hinckmar actually asserted
that when St. Remy was about to consecrate with oil, the humble
King Clovis, at his coronation, a dove descended from Heaven, and
placed in his hands a small vial of holy oil. Hinckmar defied any man
to prove the contrary. As he further declared that the vial of oil was
still to be found in the saint’s sepulchre, and as it was so found,
accordingly, Hinckmar was allowed to have proved his case.
Thenceforward, the chevaliers of the St. Ampoule were created, for a
day—that of the crowning of the sovereign. They had charge of the
vial, delivered it to the archbishop, and saw it restored to its
repository; and therewith, the coronation and their knightly character
concluded together. From that time, down to the period of Louis XVI.,
the knights and the vial formed the most distinguished portion of the
coronation procession and doings at the crowning of the kings of
France.
Then ensued the Revolution; and as that mighty engine never
touched anything without smashing it, you may be sure that the vial
of St. Remy hardly escaped destruction.
On the 6th of October, 1793, Citizen Rhull entered the modest
apartment of Philippe Hourelle, chief marguillier of the Cathedral of
Rheims, and without ceremony demanded that surrender should be
made to him of the old glass-bottle of the ci-devant Remy. Philippe’s
wig raised itself with horror; but as Citizen Rhull told him that it would
be as easy to lift his head from his shoulders as his wig from his
head, if he did not obey, the marguillier stammered out an assertion
that the reliquary was in the keeping of the curé, M. Seraine, to
whom he would make instant application.
“Bring pomatum and all,” said Citizen Rhull, who thus profanely
misnamed the sacred balm or thickened oil, which had anointed the
head and loins of so many kings from Charles the Bald, downward.
“May I ask,” said Philippe, timidly, “what you will do therewith?”
“Grease your neck, that the knife may slip the easier through it,
unless you bring it within a decade of minutes.”
“Too much honor by half,” exclaimed Philippe. “I will slip to the curé
as rapidly as if I slid the whole way on the precious ointment itself.
Meanwhile, here is a bottle of Burgundy—”
“Which I shall have finished within the time specified. So, despatch;
and let us have t’other bottle, too!”
When Philippe Hourelle had communicated the request to the curé,
Monsieur Seraine, with a quickness of thought that did justice to his
imagination, exclaimed, “We will take the rogues in, and give them a
false article for the real one.” But the time was so short; there was no
second ancient-looking vial at hand; there was not a pinch of
pomatum, nor a spoonful of oil in the house, and the curé confessed,
with a sigh, that the genuine relic must needs be surrendered. “But
we can save some of it!” cried M. Seraine; “here is the vial, give me
the consecrating spoon.” And with the handle of the spoon, having
extracted some small portions, which the curé subsequently
wrapped up carefully, and rather illegibly labelled, the vial was
delivered to Philippe, who surrendered it to Citizen Rhull, who
carried the same to the front of the finest cathedral in France, and at
the foot of the statue of Louis XV. Citizen Rhull solemnly hammered
the vial into powder, and, in the name of the Republic, trod the
precious ointment underfoot till it was not to be distinguished from
the mud with which it was mingled.
“And so do we put an end to princes and pomatum,” cried he.
Philippe coughed evasively; smiled as if he was of the same way of
thinking with the republican, and exclaimed, very mentally indeed,
“Vivent les princes et la pommade.” Neither, he felt assured, was
irrevocably destroyed.
The time, indeed, did come round again for princes, and Napoleon
was to be crowned at Notre Dame. He cared little as to what had
become of the Heaven-descended ointment, and he might have
anointed, as well as crowned, himself. There were some dozen
gentlemen who hoped that excuse might be discovered for creating
the usual order of the Knights of the Ampoule; but the Emperor did
not care a fig for knights or ointment, and, to the horror of all who
hoped to be chevaliers, the imperial coronation was celebrated
without either. But then Napoleon was discrowned, as was to be
expected from such profanity; and therewith returned the Bourbons,
who, having forgotten nothing, bethought themselves of the Saint
Ampoule. Monsieur de Chevrières, magistrate at Rheims, set about
the double work of discovery and recovery. For some time he was
unsuccessful. At length, early in 1819, the three sons of the late
Philippe Hourelle waited on him. They made oath that not only were
they aware of a portion of the sacred ointment having been in the
keeping of their late father, but that his widow succeeded the
inheritance, and that she reckoned it as among her choicest
treasures.
“She has nothing to do but to make it over to me,” said Monsieur
Chevrières; “she will be accounted of in history as the mother of the
knights of the Ampoule of the Restoration.”
“It is vexatious,” said the eldest son, “but the treasure has been lost.
At the time of the invasion, our house was plundered, and the relic
was the first thing the enemy laid his hands on.”
The disappointment that ensued was only temporary. A judge named
Lecomte soon appeared, who made oath that he had in his keeping
a certain portion of what had at first been consigned to the widow
Hourelle. The portion was so small that it required an eye of faith,
very acute and ready indeed, to discern it. The authorities looked
upon the relic, and thought if Louis XVIII. could not be crowned till a
sufficient quantity of the holy ointment was recovered wherewith to
anoint him, the coronation was not likely to be celebrated yet awhile.
Then arose a crowd of priests, monks, and ex-monks, all of whom
declared that the curé, M. Seraine, had imparted to them the secret
of his having preserved a portion of the dried anointing oil, but they
were unable to say where he had deposited it. Some months of
hesitation ensued, when, in summer, M. Bouré, a priest of Berry-au-
Bac, came forward and proclaimed that he was the depositary of the
long-lost relic, and that he had preserved it in a portion of the
winding-sheet of St. Remy himself. A week later M. Champagne
Provotian appeared, and made deposition to the following effect: He
was standing near Rhull when the latter, in October, 1793, destroyed
the vial which had been brought from Heaven by a dove, at the foot
of the statue of Louis XV. When the republican struck the vial, some
fragments of the glass flew on to the coat-sleeve of the said M.
Champagne. These he dexterously preserved, took home with him,
and now produced in court.
A commission examined the various relics, and the fragments of
glass. The whole was pronounced genuine, and the chairman
thought that by process of putting “that and that together,” there was
enough of legend, vial, and ointment to legitimately anoint and
satisfy any Christian king.
“There is nothing now to obstruct your majesty’s coronation,” said his
varlet to him one morning, after having spent three hours in a service
for which he hoped to be appointed one of the knights of the Sainte
Ampoule; “there is now absolutely nothing to prevent that august
ceremony.”
“Allons donc!” said Louis XVIII. with that laugh of incredulity, that
shrug of the shoulders, and that good-humored impatience at
legends and absurdities, which made the priests speak of him as an
infidel.
“What shall be done with the ointment?” said the knight-expectant.
“Lock it up in the vestry cupboard, and say no more about it.” And
this was done with some ceremony and a feeling of disappointment.
The gathered relics, placed in a silver reliquary lined with white silk,
and enclosed in a metal case under three locks, were deposited
within the tomb of St. Remy. There it remained till Charles X. was
solemnly crowned in 1825. In that year, positively for the last time,
the knights of the Sainte Ampoule were solemnly created, and did
their office. As soon as Charles entered the choir, he knelt in the
front of the altar. On rising, he was led into the centre of the
sanctuary, where a throned chair received his august person. A
splendid group half-encircled him; and then approached the knights
of the Sainte Ampoule in grand procession, bearing all that was left
of what the sacred dove did or did not bring to St. Remy, for the
anointing of Clovis. Not less than three prelates, an archbishop and
two bishops, received the ointment from the hands of the knights,
and carried it to the high altar. Their excellencies and eminences
may be said to have performed their office with unction, but the
people laughed alike at the knights, the pomatum, and the
ceremony, all of which combined could not endow Charles X. with
sense enough to keep his place. The knights of the Sainte Ampoule
may be said now to have lost their occupation for ever.
Of all the memorabilia of Rheims, the good people there dwelt upon
none more strongly than the old and splendid procession of these
knights of the Sainte Ampoule. The coronation cortège seemed only
a subordinate point of the proceedings; and the magnificent canopy,
upheld by the knights over the vial, on its way from the abbey of St.
Remy to the cathedral, excited as much attention as the king’s
crown.
The proceedings, however, were not always of a peaceable
character. The Grand Prior of St. Remy was always the bearer of the
vial, in its case or shrine. It hung from his neck by a golden chain,
and he himself was mounted on a white horse. On placing the vial in
the hands of the archbishop, the latter pledged himself by solemn
oath to restore it at the conclusion of the ceremony; and some half-
dozen barons were given as hostages by way of security. The
procession back to the abbey, through the gayly tapestried streets,
was of equal splendor with that to the cathedral.
The horse on which the Grand Prior was mounted was furnished by
the government, but the Prior claimed it as the property of the abbey
as soon as he returned thither. This claim was disputed by the
inhabitants of Chêne la Populeux, or as it is vulgarly called, “Chêne
la Pouilleux.” They founded their claim upon a privilege granted to
their ancestors. It appeared that in the olden time, the English had
taken Rheims, plundered the city, and rifled the tomb of St. Remy,
from which they carried off the Sainte Ampoule. The inhabitants of
Chêne, however, had fallen upon the invaders and recovered the
inestimable treasure. From that time, and in memory and
acknowledgment of the deed, they had enjoyed, as they said, the
right to walk in the procession with the knights of the Sainte
Ampoule, and had been permitted to claim the horse ridden by the
Grand Prior. The Prior and his people called these claimants scurvy
knaves, and would by no means attach any credit to the story. At the
coronation of Louis XIII. they did not scruple to support their claim by
violence. They pulled the Prior from his horse, terribly thrashed the
monks who came to his assistance, tore the canopy to pieces,
thwacked the knights right lustily, and carried off the steed in triumph.
The respective parties immediately went to law, and spent the value
of a dozen steeds, in disputes about the possession of a single
horse. The contest was decided in favor of the religious community;
and the turbulent people of Chêne were compelled to lead the
quadruped back to the abbey stables. They renewed their old claim
subsequently, and again threatened violence, much to the delight of
the attorneys, who thought to make money by the dissension. At the
coronations of Louis XV. and Louis XVI. these sovereigns issued
special decrees, whereby the people of Chêne were prohibited from
pretending to any property in the horse, and from supporting any
such pretensions by acts of violence.
The history of foreign orders would require a volume as large as
Anstis’s; but though I can not include such a history among my
gossiping details, I may mention a few curious incidents connected
with
THE ORDER OF THE HOLY GHOST.
There is a singular circumstance connected with this order. It was
founded by the last of the Valois, and went out with the last of the
Bourbons. Louis Philippe had a particular aversion for the orders
which were most cherished by the dynasty he so cleverly
supplanted. The Citizen King may be said to have put down both “St.
Louis” and the “Holy Ghost” cavaliers. He did not abolish the orders
by decree; but it was clearly understood that no one wearing the
insignia would be welcome at the Tuileries.
The Order of the Holy Ghost was instituted by Henri, out of gratitude
for two events, for which no other individual had cause to be grateful.
He was (when Duke of Anjou) elected King of Poland, on the day of
Pentecost, 1573, and on the same day in the following year he
succeeded to the crown of France. Hence the Order with its hundred
members, and the king as grand master.
St. Foix, in his voluminous history of the order, furnishes the
villanous royal founder with a tolerably good character. This is more
than any other historian has done; and it is not very satisfactorily
executed by this historian himself. He rests upon the principle that
the character of a king, or his disposition rather, may be judged by
his favorites. He then points to La Marck, Mangiron, Joyeuse,
D’Epernon, and others. Their reputations are not of the best, rather
of the very worst; but then St. Foix says that they were all admirable
swordsmen, and carried scars about them, in front, in proof of their
valor: he evidently thinks that the bellica virtus is the same thing as
the other virtues.
On the original roll of knights there are names now more worthy of
being remembered. Louis de Gonzague, Duke de Nevers, was one
of these. On one occasion, he unhorsed the Huguenot Captain de
Beaumont, who, as he lay on the ground, fired a pistol and broke the
ducal kneepan. The Duke’s squire bent forward with his knife to
despatch the Captain; the Duke, however, told the latter to rise. “I
wish,” said he, “that you may have a tale to tell that is worth
narrating. When you recount, at your fireside, how you wounded the
Duke de Nevers, be kind enough to add that he gave you your life.”
The Duke was a noble fellow. Would that his generosity could have
restored his kneepan! but he limped to the end of his days.
But there was a nobler than he, in the person of the Baron d’Assier,
subsequently Count de Crussol and Duke d’Uzes. He was a
Huguenot, and I confess that I can not account for the fact of his
being, at any time of his life, a Knight of the Order of the Holy Ghost.
Henri III. was not likely to have conferred the insignia even on a
pervert. His name, however, is on the roll. He was brave, merciful,
pious, and scrupulously honest. When he captured Bergerai, he
spared all who had no arms in their hands, and finding the women
locked up in the churches, he induced them to return home, on
promise of being protected from all molestation. These poor
creatures must have been marvellously fair; and the baron’s eulogy
on them reminds me of the expression of the soldiers when they led
Judith through the camp of Holofernes: “Who could despise this
people that have among them such women.”
The baron was not a little proud of his feat, and he thought that if all
the world talked of the continence of Scipio, he had a right to claim
some praise as the protector of female virtue. Accordingly, in
forwarding an account of the whole affair to the Duc de Montpensier,
he forwarded also a few samples of the ladies. “I have only chosen
twenty of the handsomest of them,” he writes, “whom I have sent you
that you may judge if they were not very likely to tempt us to
reprisals; they will inform you that they have suffered not the least
dishonor.” By sending them to Montpensier’s quarters the ladies
were in great danger of incurring that from which the Baron had
saved them. But he winds up with a small lecture. He writes to the
Duke: “You are a devotee [!]; you have a ghostly father; your table is
always filled with monks; your hear two or three masses every day;
and you go frequently to confession. I confess myself only to God. I
hear no masses. I have none but soldiers at my table. Honor is the
sole director of my conscience. It will never advise me to order
violence against woman, to put to death a defenceless enemy, or to
break a promise once given.” In this lecture, there was, in fact, a
double-handed blow. Two birds were killed with one stone. The
Baron censured, by implication, both the Duke and his religion. I was
reminded of him by reading a review in the “Guardian,” where the
same skilful method is applied to criticism. The reviewer’s subject
was Canon Wordsworth’s volume on Chevalier Bunsen’s
“Hippolytus.” “The canon’s book,” said the reviewer (I am quoting
from memory), “reminds us—and it must be a humiliation and
degradation to an intelligent, educated, and thoughtful man—of one
of Dr. Cumming’s Exeter Hall lectures.” Here the ultra high church
critic stunned, with one blow, the merely high-church priest and the
no-church presbyterian.
There was generosity, at least, in another knight of this order,
Francis Goaffier, Lord of Crèvecœur. Catherine of Medicis
announced to him the appointment of his son to the command of a
regiment of foot. “Madame,” said the Knight of the Holy Ghost, “my
son was beset, a night or two ago, by five assassins; a Captain La
Vergne drew in his defence, and slew two of the assailants. The rest
fled, disabled. If your majesty will confer the regiment on one who
deserves it, you will give it to La Vergne.”—“Be it so,” said Catherine,
“and your son shall not be the less well provided for.”
One, at least, of the original knights of this order was famous for his
misfortunes; this was Charles de Hallewin, Lord of Piennes. He had
been in six-and-twenty sieges and battles, and never came out of
one unscathed. His domestic wounds were greater still. He had five
sons, and one daughter who was married. The whole of them, with
his son-in-law, were assassinated, or died accidentally, by violent
deaths. The old chevalier went down to his tomb heart-broken and
heirless.
Le Roi, Lord of Chavigny, and who must not be mistaken for an
ancestor of that Le Roi who died at the Alma under the title of
Marshal St. Arnaud, is a good illustration of the blunt, honest knight.
Charles IX. once remarked to him that his mother, Catherine de
Medicis, boasted that there was not a man in France, with ten
thousand livres a year, at whose hearth she had not a spy in her pay.
“I do not know,” said Le Roi, “whether tyrants make spies, or spies
tyrants. For my own part, I see no use in them, except in war.”
For honesty of a still higher sort, commend me to Scipio de Fierques,
Lord of Lavagne. Catherine de Medicis offered to make this, her
distant relative, a marshal of France. “Good Heavens, Madame!” he
exclaimed, “the world would laugh at both of us. I am simply a brave
gentleman, and deserve that reputation; but I should perhaps lose it,
were you to make a marshal of me.” The dignity is taken with less
reluctance in our days. It was this honest knight who was asked to
procure the appointment of queen’s chaplain for a person who, by
way of bribe, presented the gallant Scipio with two documents which
would enable him to win a lawsuit he was then carrying on against
an obstinate adversary. Scipio perused the documents, saw that they
proved his antagonist to be in the right, and immediately withdrew
his opposition. He left the candidate for the queen’s chaplaincy to
accomplish the object he had in view, in the best way he might.
There was wit, too, as might be expected, among these knights.
John Blosset, Baron de Torci, affords us an illustration. He had been
accused of holding correspondence with the enemy in Spain, and
report said that he was unworthy of the Order of the Holy Ghost. He
proved his innocence before a chapter of the order. At the end of the
investigation, he wittily applied two passages from the prayer-book of
the knights, by turning to the king, and saying, “Domine ne projicias
me a facie tuâ, et spiritum sanctum tuum ne auferas a me.” “Lord,
cast me out from thy presence, and take not thy ‘Holy Spirit’ from
me.” And the king bade him keep it, while he laughed at the rather
profane wit of John Blosset.
There was wit, too, of a more practical nature, among these knights
of the Holy Spirit. The royal founder used occasionally to retire with
the knights to Vincennes. There they shut themselves up, as they
said, to fast and repent; but, as the world said, to indulge in
pleasures of a very monster-like quality. The royal dukes of a later
period in France used to atone for inordinate vice by making their
mistresses fast; the royal duchesses settled their little balance with
Heaven, by making their servants fast. It appears that there was
nothing of this vicarious penance in the case of Henri III. and his
knights. Not that all the knights willingly submitted to penance which
mortified their appetites. Charles de la Marck, Count of Braine. was
one of those impatient penitents. On a day on which rigid abstinence
had been enjoined, the king was passing by the count’s apartment,
when he was struck by a savory smell. King as he was, he
immediately applied his eye to the keyhole of the count’s door, and
beheld the knight blowing lustily at a little fire under a chafing-dish, in
which there were two superb soles frying in savory sauce. “Brother
knight, brother knight,” exclaimed Henri, “I see all and smell much.
Art thou not ashamed thus to transgress the holy rule?”—“I should
be much more so,” said the count, opening the door, “if I made an
enemy of my stomach. I can bear this sort of abstinence no longer.
Here am I, knight and gentleman, doubly famished in that double
character, and I have been, in my own proper person, to buy these
soles, and purchase what was necessary for the most delicious of
sauces: I am cooking them myself, and they are now done to a turn.
Cooked aux gratins, your majesty yourself can not surely resist
tasting. Allow me”—and he pushed forward a chair, in which Henri
seated himself, and to the “soles aux gratins,” such as Vefour and
Very never dished up, the monarch sat down, and with the hungry
count, discussed the merits of fasting, while they enjoyed the fish. It
was but meagre fare after all; and probably the repast did not
conclude there.
Charity is illustrated in the valiant William Pot (a very ancient name
of a very ancient family, of which the late archdeacon of Middlesex
and vicar of Kensington was probably a descendant). He applied a
legacy of sixty thousand livres to the support of wounded soldiers.
Henri III., who was always intending to accomplish some good deed,
resolved to erect an asylum for infirm military men; but, of course, he
forgot it. Henri IV., who has received a great deal more praise than
he deserves, also expressed his intention to do something for his old
soldiers; but he was too much taken up with the fair Gabrielle, and
she was not like Nell Gwynne, who turned her intimacy with a king to
the profit of the men who poured out their blood for him. The old
soldiers were again neglected; and it was not till the reign of Louis
XIV. that Pot’s example was again recalled to mind, and profitable
action adopted in consequence. When I think of the gallant Pot’s
legacy, what he did therewith, and how French soldiers benefited
thereby, I am inclined to believe that the German troops, less well
cared for, may thence have derived their once favorite oath, and that
Potz tausend! may have some reference to the sixty thousand livres
which the compassionate knight of Rhodes and the Holy Ghost
devoted to the comfort and solace of the brave men who had been
illustriously maimed in war.
The kings of France were accustomed to create a batch of knights of
the Holy Ghost, on the day following that of the coronation, when the
monarchs became sovereign heads of the order. The entire body
subsequently repaired from the Cathedral to the Church of St. Remi,
in grand equestrian procession, known as the “cavalcade.” Nothing
could well exceed the splendor of this procession, when kings were
despotic in France, and funds easily provided. Cavalry and infantry
in state uniforms, saucy pages in a flutter of feathers and ribands,
and groups of gorgeous officials preceded the marshals of France,
who were followed by the knights of the Holy Ghost, after whom rode
their royal Grand Master, glittering like an Eastern king, and nodding,
as he rode, like a Mandarin.
The king and the knights performed their devotions before the shrine
of Saint Marcoul, which was brought expressly from the church of
Corbeni, six leagues distance from Rheims. This particular ceremony
was in honor of the celebrated old abbot of Nantua, who, in his
lifetime, had been eminently famous for his success in curing the
scrofulous disorder called “the king’s evil.” After this devotional
service, the sovereign master of the order of the Holy Ghost was
deemed qualified to cure the evil himself. Accordingly, decked with
the mantle and collar of the order, and half encircled by the knights,
he repaired to the Abbey Park to touch and cure those who were
afflicted with the disease in question. It was no little labor. When
Louis XVI. performed the ceremony, he touched two thousand four
hundred persons. The form of proceeding was singular enough. The
king’s first physician placed his hand on the head of the patient;
upon which a captain of the guard immediately seized and held the
patient’s hands closely joined together. The king then advanced,
head uncovered, with his knights, and touched the sufferers. He
passed his right hand from the forehead to the chin, and from one
cheek to the other; thus making the sign of the cross, and at the
same time pronouncing the words, “May God cure thee; the king
touches thee!”
In connection with this subject, I may add here that Evelyn, in his
diary, records that Charles II. “began first to touch for the evil,
according to custom,” on the 6th of July, 1660, and after this fashion.
“His Majesty sitting under his state in the Banqueting House, the
chirurgeons caused the sick to be brought, and led up to the throne,
where they kneeling, the king strokes their faces or cheeks with both
his hands at once, at which instant a chaplain, in his formalities,
says, ‘He put his hands upon them, and He healed them.’ This is
said to every one in particular. When they have been all touched,
they come up again in the same order, and the other chaplain
kneeling, and having angel-gold strung on white riband on his arm,
delivers them one by one to his Majesty, who puts them about the
necks of the touched as they pass, while the first chaplain repeats,
‘That is the true light who came into the world.’” The French
ceremonial seems to me to have been the less pretentious; for the
words uttered by the royal head of the order of the Holy Ghost,
simply formed a prayer, and an assertion of a fact: “May God heal
thee; the king touches thee!” And yet who can doubt the efficacy of
the royal hand of Charles II., seeing that, at a single touch, he not
only cured a scrofulous Quaker, but converted him into a good
churchman?
The history of the last individual knight given in these imperfect
pages (Guy of Warwick), showed how history and romance wove
themselves together in biography. Coming down to a later period, we
may find another individual history, that may serve to illustrate the
object I have in view. The Chevalier de Bayard stands prominently
forward. But there was before his time, a knight who was saluted by
nearly the same distinctive titles which were awarded to Bayard. I
allude to Jacques de Lelaing, known as “the knight without fear and
without doubt.” His history is less familiar to us, and will, therefore,
the better bear telling. Besides, Bayard was but a butcher. If he is not
to be so accounted, then tell us, gentle shade of Don Alonzo di
Sotomayor, why thy painful spirit perambulates the groves of
Elysium, with a scented handkerchief alternately applied to the hole
in thy throat and the gash in thy face? Is it not that, with cruel
subtlety of fence Bayard run his rapier into thy neck “four good
finger-breadths,” and when thou wast past resistance, did he not
thrust his dagger into thy nostrils, crying the while, “Yield thee,
Signor Alonzo, or thou diest!” The shade of the slashed Spaniard
bows its head in mournful acquiescence, and a faint sound seems to
float to us upon the air, out of which we distinguish an echo of “The
field of Monervyne.”
JACQUES DE LELAING,
THE GOOD KNIGHT WITHOUT FEAR AND WITHOUT DOUBT.
“Faites silence; je vais parler de lui!”— Boileau.
Between the city of Namur and the quaint old town of Dinant
there is as much matter of interest for the historian as of beauty for
the traveller and artist. War has been the most terrible scourge of the
two localities on the Meuse which I have just named. Namur has a
present reputation for cutlery, and an old one for “slashing blades” of
another description. Don John, the great victor at Lepanto, lies
entombed in the city, victim of the poison and the jealousy of his
brother Philip. There the great Louis proved himself a better soldier
than Boileau did a poet, when he attempted to put the royal soldier’s
deeds into rhyme. Who, too, can stand at St. Nicholas’s gate, without
thinking of “my uncle Toby,” and the Frenchmen, for whose dying he
cared so little, on the glacis of Namur? At present the place, it is true,
has but a dull and dreamy aspect. Indeed, it may be said of the
inhabitants, as of Molly Carew’s lovers, that “It’s dhrames and not
sleep that comes into their heads.” Such, at least, would seem to be
the case, if I may draw a conclusion from what I saw during the last
summer, at the bookseller’s stall at the Namur station, where I found
more copies of a work professing to interpret dreams than of any
other production, whether grave or gaillard.
Dinant, a curious old town, the high limestone-rocks behind which
seems to be pushing it from off its narrow standing-ground into the
Meuse, has even bloodier reminiscences than Namur; but of these I
will not now speak. Between the two cities, at the most picturesque
part of the stream, and on the loftiest cliff which rises above the
stream, is the vast ruin of the old titanic castle of Poilvache, the once
rather noisy home of the turbulent household of those terrible
brothers, known in chivalrous history as the “Four Sons of Aymon.”
During one of the few fine evenings of the last summer, I was looking
up at this height, from the opposite bank, while around me stood in
groups a number of those brilliant-eyed, soft-voiced, ready-witted
Walloons, who are said to be the descendants of a Roman legion,
whose members colonized the country and married the ladies in it! A
Walloon priest, or one at least who spoke the dialect perfectly, but
who had a strong Flemish accent when addressing to me an
observation in French, remained during the period of my observation
close at my side. “Are these people,” said I to him, “a contented
people?” He beckoned to a cheerful-looking old man, and assuming
that he was contented with the dispensation that had appointed him
to be a laborer, inquired of him which part of his labor he loved best?
After pausing for a minute, the old peasant replied in very fair
French, “I think the sweetest task I have is when I mow that meadow
up at Bloquemont yonder, for the wild thyme in it embalms the very
air.” “But your winter-time,” said I, “must be a dark and dreary time.”
“Neither dark nor dreary,” was the remark of a tidy woman, his wife,
who was, at the moment, on her knees, sewing up the ragged rents
in the gaberdine of a Walloon beggar—“Neither dark nor dreary. In
winter-time, at home, we don’t want light to get the children about us
to teach them their catechism.” The priest smiled. “And as for spring-
time,” said her husband, “you should be here to enjoy it; for the fields
are then all flower, and the sky is one song.” “There is poetry in their
expressions,” said I to the priest. “There is better than that,” said he,
“there is love in their hearts;” and, turning to the woman who was
mending the raiment of the passive mendicant, he asked her if she
were not afraid of infection. “Why should I fear?” was her remark. “I
am doing but little; Christ did more; He washed the feet of beggars;
and we must risk something, if we would gain Paradise.” The
particular beggar to whom she was thus extending most practical
charity was by no means a picturesque bedesman; but, not to be
behind-hand in Χάρις toward him, I expressed compassion for his lot.
“My lot is not so deplorable,” said he, uncovering his head; “I have
God for my hope, and the charity of humane people for my succor.”
As he said this, my eye turned from him to a shepherd who had just
joined our group, and who was waiting to be ferried over to the little
village of Houx. I knew him by name, and knew something of the
solitariness of his life, and I observed to him, “Jacques, you, at least,
have a dull life of it; and you even now look weary with the long

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Bacteriophage Therapy From Lab to

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For further volumes:

From Lab to Clinical Practice

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media LLC 2018

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

Braga, Portugal Joana Azeredo

PART I ISOLATION OF BACTERIOPHAGES

PART II CHARACTERIZATION OF BACTERIOPHAGES

4 In Vitro Activity of Bacteriophages Against Planktonic and

PART III BACTERIOPHAGE SELECTION AND COCKTAIL FORMULATION

7 Determination of the Bacteriophage Host Range:

PART IV BIODISTRIBUTION, HOST INTERACTION AND CLINICAL APPLICATION

12 Interaction of Bacteriophages with the Immune System:

PART V PRODUCTION, PURIFICATION AND BACTERIOPHAGE STORAGE

15 Bacteriophage Production in Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

PART VI SAFETY AND REGULATION

18 Bacteriophage Production in Compliance with

PART VII NEW PHAGE THERAPY APPROACHES

20 Nano/Micro Formulations for Bacteriophage Delivery . . . . . . . . . . . . . . . . . . . . . . 271

MARYAM AGBOLUAJE  Chemical and Materials Engineering, University of Alberta,

VERA V. MOROZOVA  Laboratory of Molecular Microbiology, Institute of Chemical Biology

Isolation of Bacteriophages for Fastidious Bacteria

Helicobacter pylori is a Gram-negative, spiral, and microaerophilic

lost pseudolysogenic ability via spontaneous mutations may also be

2.1 Bacteriophage 1. H. pylori strains.

2.2 Purification 1. BCV: Brucella broth including 0.5% (wt/vol) β-cyclodextrin

7. CsCl (ρ ¼ 1.7, 1.5, and ρ ¼ 1.3) in AAS solution.

This protocol shows a detailed description of the method previ-

3.1 Preparation 1. H. pylori strains are shaken vigorously in 2 or 5 mL BEV in

3.3 Isolation 1. Filtrates in which the presence of bacteriophages has been

10. The purified bacteriophage sample can be used for electronic

1. In the preparation of 1000 mL, 900 mL of water including

6. H. pylori strains seem commonly to have strong restriction-

This work was supported by Grants-in-Aid for Scientific Research

Isolation of Bacteriophages of the Anaerobic Bacteria

A number of bacteriophages have been incorporated in several

Fig. 1 Typical lytic plaques of phages infecting Bacteroides fragilis grown on

In this chapter, we report a methodology for the detection and

2.1 Handling 1. Bacteroides fragilis RYC2056 [10], although other Bacteroides

L-cystein, 1.8 g glucose, 0.12 g MgSO4·7H2O, and add

through a 0.2 μm pore size high protein binding membrane

(vol/vol)). Pour into Petri dishes (20 mL in dishes of 9 cm

2.2 Host Strain Stock 1. Bacteroides host strain.

2.3 Preparation 1. Bacteroides host strain stock culture.

2.4 Bacteriophage 1. Bacteroides host strain inoculum culture.

6. Anaerobic jar or chamber.

2.5 Bacteriophage 1. Bacteroides host strain inoculum culture.

2.6 Isolation 1. Bacteriophage plaques.

2.7 Preparation of a 1. Bacteriophage suspension.

12. Anaerobic jar or chamber.

2.8 Preparation 1. Reference bacteriophage: Bacteriophage B56-3 (ATCC

3.4 Bacteriophage 1. Add 1 mL of the inoculum culture to 2.5 mL of melted

3.5 Bacteriophage 1. Add 1 mL of the inoculum culture grown at OD620 0.3–0.5,

MARYAM AGBOLUAJE Chemical and Materials Engineering, University of Alberta,

VERA V. MOROZOVA Laboratory of Molecular Microbiology, Institute of Chemical Biology