![At the word “Man both sides,” each watch will repair to its
respective side, the odd numbers standing to the left of the left guns;
even numbers to the right of the right guns.
“Man both sides.”
Left guns.—No. 3 remains 3; 5 becomes 4; 7—6; 9—5; 11—2; 13—
7; 1 remains 1.
Right guns.—No. 4 remains 4; 6 becomes 3; 8—6; 10—5; 12—2; 2
—1.
Note.—The left guns are odd starboard, and even port. The right
guns are even starboard, and odd port. The odd numbered guns’
crews are taken from the starboard watch; the even numbered from
the port watch.
Guns’ crews always man, and powder boys always supply
adjacent guns, when clearing for action, or when fighting both sides.
Note.—With a crew of 11 men, and upwards, and both sides
manned, No. 2 is always to attend the train tackle.
When casualties occur at the guns, those holding the highest
numbers, or those last placed, will be the first to move to fill up the
vacancies, excepting that where both captains are removed, the
officer will name the most fitting person to become No. 1, filling up
the vacancy as above. For instance; if there should be 13 men at the
gun, and Nos. 3, 6, and 9, are ordered to “fall out,” Nos. 5, and 7,
move up, becoming Nos. 3, and 5; No. 11, moves up, and becomes
No. 7; No. 13,—No. 9; No. 8 moves up, and becomes No. 6; No. 10,
—No. 8; and No. 12,—No. 10.[32]
Exercise with 7 Men, to a 32-pounder carronade.
No.
1. The Captain; commands, attends the breeching, primes, points,
fires, and stops the vent.
2. The second Captain; assists No. 1, casts loose, runs out, attends
the apron, and elevating screw, cocks the lock, and attends](https://image.slidesharecdn.com/57922-250617195159-53691dbf/85/Candida-albicans-Methods-and-Protocols-1st-Edition-T-Sreevalsan-Auth-33-320.jpg)
![“Man both sides.”—“Prime.”—“Point.”—“Muzzle to the
right.”—“Muzzle to the left.”—“Elevate.”—“Ready.”—(Left guns.)
—“Fire.”—“Sponge, and load.”—(Right guns.)—“Fire.”—“Sponge,
and load.”—(Left guns.)—“Run out.”—“Fire.”—“Sponge, and load.”—
(Right guns.)—“Run out.”—“Fire.”—“Cease firing.”
When ordered to “cease firing,” the guns are to be loaded, and run
out.[33]
Exercise for the 10-inch, or other revolving Gun, with a crew
of 17 men.
The crew are assembled as in the established Gun exercise; then
—
Gun.—Nos. 1, 2, 3, 4, 5, 6.
Auxiliaries.—Nos. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.
Traversing tackle-men.—Nos. 7, 8.
Handspike-men.—Nos. 9, 10. Assistant handspike-men.—
Nos. 11, 12.
Compressor men, Nos. 13, 14.
Rear-men.—No. 16, the right rear-man. No. 17, the left rear-
man.
No. 1. The Captain. No. 4. Sponger.
” 2. Second Captain. ” 5. Assistant loader.
” 3. Loader. ” 6. Assistant sponger.
Words of command.
“Traverse.”[34]
—“Prime.”—“Point.”—“Elevate.”—“Ready.”—“Fire.”— “Stop the
vent.”—“Sponge.”—“Load.”—“Run out.”
Exercise with 17 men.
No.](https://image.slidesharecdn.com/57922-250617195159-53691dbf/85/Candida-albicans-Methods-and-Protocols-1st-Edition-T-Sreevalsan-Auth-36-320.jpg)
![D = Number in each case.
E = Number of cases.
50 Gun Frigate.
Nature of
Ordnance.
A Charge. B C D E Spare powder.
8 in. Gun
65 cwt.
8
Reduced
5 lb.
Full
8 lb.
Distant
10 lb.
20 160 15 11
20 rounds per
gun,
reduced
charges, as
spare;
amounting to
800 lb.
40 320 14 23
20 160 11 15
32 Pr.
56 cwt.
22
Reduced
6 lb.
Full
8 lb.
Distant
10 lb.
40 880 19 46
20 rounds per
gun,
reduced
charges, as
spare;
amounting to
2640 lb.
20 440 14 32
20 440 11 40
32 Pr.
45 cwt.
20
Reduced
5 lb.
Full
7 lb.
30 600 22 27½
20 rounds per
gun,
reduced
charges, as
spare;
amounting to
2000 lb.
50 1000 16 62½
24 Pr.
Howitzer
12½ cwt.
2 2½ lb. 174 348 20* 17†
2 half-cases of
bursters, (10
oz., 6 oz.,
5½ oz., and
4½ oz.,) and
two half-
cases of
fuses.
12 Pr.
Howitzer
6 cwt.
2 1¼ lb. 174 348 40* 9‡
* In a half-case. † Half-cases, and a quarter-case. ‡ Half-
cases.
ON NAVAL BOMBARDMENTS.[35]
“The attack of fortresses, and powerful land batteries with a naval
force only, must ever be a hazardous, and perhaps desperate
undertaking. But if skilfully combined with a military force sufficiently
strong to make good its landing, to invest the place, or the batteries](https://image.slidesharecdn.com/57922-250617195159-53691dbf/85/Candida-albicans-Methods-and-Protocols-1st-Edition-T-Sreevalsan-Auth-43-320.jpg)
Candida albicans Methods and Protocols 1st Edition T. Sreevalsan (Auth.)
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- 5. Candida albicans Methods and Protocols 1st Edition T. Sreevalsan (Auth.) Digital Instant Download Author(s): T. Sreevalsan (auth.), Ronald L. Cihlar, Richard A. Calderone (eds.) ISBN(s): 9781588297600, 1588297608 Edition: 1 File Details: PDF, 2.25 MB Year: 2009 Language: english
- 8. M E T H O D S I N M O L E C U L A R B I O L O G Y TM John M. Walker, SERIES EDITOR 502. Bacteriophages: Methods and Protocols, Volume 2: Molecular and Applied Aspects, edited by Martha R. J. Clokie and Andrew M. Kropinski 2009 501. Bacteriophages: Methods and Protocols, Volume 1: Isolation, Characterization, and Interactions, edited by Martha R. J. Clokie and Andrew M. Kropinski 2009 499. Candida albicans: Methods and Protocols, edited by Ronald L. Cihlar and Richard A. Calderone, 2009 496. DNA and RNA Profiling in Human Blood: Methods and Protocols, edited by Peter Bugert, 2009 493. Auditory and Vestibular Research: Methods and Protocols, edited by Bernd Sokolowski, 2009 490. Protein Structures, Stability, and Interactions, edited by John W. Schriver, 2009 489. Dynamic Brain Imaging: Methods and Protocols, edited by Fahmeed Hyder, 2009 485. HIV Protocols: Methods and Protocols, edited by Vinayaka R. Prasad and Ganjam V. Kalpana, 2009 484. Functional Proteomics: Methods and Protocols, edited by Julie D. Thompson, Christine Schaeffer-Reiss, and Marius Ueffing, 2008 483. Recombinant Proteins From Plants: Methods and Protocols, edited by Loı̈c Faye and Veronique Gomord, 2008 482. Stem Cells in Regenerative Medicine: Methods and Protocols, edited by Julie Audet and William L. Stanford, 2008 481. Hepatocyte Transplantation: Methods and Protocols, edited by Anil Dhawan and Robin D. Hughes, 2008 480. Macromolecular Drug Delivery: Methods and Protocols, edited by Mattias Belting, 2008 479. Plant Signal Transduction: Methods and Protocols, edited by Thomas Pfannschmidt, 2008 478. Transgenic Wheat, Barley and Oats: Production and Characterization Protocols, edited by Huw D. Jones and Peter R. Shewry, 2008 477. Advanced Protocols in Oxidative Stress I, edited by Donald Armstrong, 2008 476. Redox-Mediated Signal Transduction: Methods and Protocols, edited by John T. Hancock, 2008 475. Cell Fusion: Overviews and Methods, edited by Elizabeth H. Chen, 2008 474. Nanostructure Design: Methods and Protocols, edited by Ehud Gazit and Ruth Nussinov, 2008 473. Clinical Epidemiology: Practice and Methods, edited by Patrick Parfrey and Brendon Barrett, 2008 472. Cancer Epidemiology, Volume 2: Modifiable Factors, edited by Mukesh Verma, 2008 471. Cancer Epidemiology, Volume 1: Host Susceptibility Factors, edited by Mukesh Verma, 2008 470. Host-Pathogen Interactions: Methods and Protocols, edited by Steffen Rupp and Kai Sohn, 2008 469. Wnt Signaling, Volume 2: Pathway Models, edited by Elizabeth Vincan, 2008 468. Wnt Signaling, Volume 1: Pathway Methods and Mammalian Models, edited by Elizabeth Vincan, 2008 467. Angiogenesis Protocols: Second Edition, edited by Stewart Martin and Cliff Murray, 2008 466. Kidney Research: Experimental Protocols, edited by Tim D. Hewitson and Gavin J. Becker, 2008 465. Mycobacteria, Second Edition, edited by Tanya Parish and Amanda Claire Brown, 2008 464. The Nucleus, Volume 2: Physical Properties and Imaging Methods, edited by Ronald Hancock, 2008 463. The Nucleus, Volume 1: Nuclei and Subnuclear Components, edited by Ronald Hancock, 2008 462. Lipid Signaling Protocols, edited by Banafshe Larijani, Rudiger Woscholski, and Colin A. Rosser, 2008 461. Molecular Embryology: Methods and Protocols, Second Edition, edited by Paul Sharpe and Ivor Mason, 2008 460. Essential Concepts in Toxicogenomics, edited by Donna L. Mendrick and William B. Mattes, 2008 459. Prion Protein Protocols, edited by Andrew F. Hill, 2008 458. Artificial Neural Networks: Methods and Applications, edited by David S. Livingstone, 2008 457. Membrane Trafficking, edited by Ales Vancura, 2008 456. Adipose Tissue Protocols, Second Edition, edited by Kaiping Yang, 2008 455. Osteoporosis, edited by Jennifer J.Westendorf, 2008 454. SARS- and Other Coronaviruses: Laboratory Protocols, edited by Dave Cavanagh, 2008 453. Bioinformatics, Volume 2: Structure, Function, and Applications, edited by Jonathan M. Keith, 2008 452. Bioinformatics, Volume 1: Data, Sequence Analysis, and Evolution, edited by Jonathan M. Keith, 2008 451. Plant Virology Protocols: From Viral Sequence to Protein Function, edited by Gary Foster, Elisabeth Johansen, Yiguo Hong, and Peter Nagy, 2008 450. Germline Stem Cells, edited by Steven X. Hou and Shree Ram Singh, 2008 449. Mesenchymal Stem Cells: Methods and Protocols, edited by Darwin J. Prockop, Douglas G. Phinney, and Bruce A. Brunnell, 2008 448. Pharmacogenomics in Drug Discovery and Develop- ment, edited by Qing Yan, 2008 447. Alcohol: Methods and Protocols, edited by Laura E. Nagy, 2008 446. Post-translational Modifications of Proteins: Tools for Functional Proteomics, Second Edition, edited by Christoph Kannicht, 2008 445. Autophagosome and Phagosome, edited by Vojo Deretic, 2008 444. Prenatal Diagnosis, edited by Sinhue Hahn and Laird G. Jackson, 2008 443. Molecular Modeling of Proteins, edited by Andreas Kukol, 2008
- 9. M E T H O D S I N M O L E C U L A R B I O L O G Y TM Candida albicans Methods and Protocols Edited by Ronald L. Cihlar and Richard A. Calderone Georgetown University Medical Center, School of Medicine, Department of Microbiology & Immunology, Washington, DC, USA
- 10. Editors Ronald L. Cihlar Georgetown University Medical Center School of Medicine Department of Microbiology & Immunology Washington DC, USA cihlarr@georgetown.edu Richard A. Calderone Georgetown University Medical Center School of Medicine Department of Microbiology & Immunology Washington DC, USA calderor@georgetown.edu Series Editor John M. Walker University of Hertfordshire Hatfield, Herts. UK ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-58829-760-0 e-ISBN 978-1-60327-151-6 DOI 10.1007/978-1-60327-151-6 Library of Congress Control Number: 2008942152 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper springer.com
- 11. Preface This book is designed to serve researchers as a source book for methodologies related to the study of medically important fungi and Candida spp., in particular. We have followed the organization of previous volumes in this series in regard to the presenta- tion of each chapter. The past decade has witnessed numerous advances in the study of human pathogenic fungi in areas of biochemistry, molecular biology, taxonomy, and physiology. In addition, the availability of genome sequences of pathogens such as Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and other model fungi has resulted in new, exciting insights into the pathogenesis of fungal diseases Thus, chapter contributions in this volume have been selected to provide the reader with a variety of approaches that cross discipline lines. For example, because of the critical importance of molecular methods, we have included chapters on reporter gene assays, transformation, gene expression in vivo, and methods for large-scale gene disruption. Chapters concerning preparation of samples for proteomic investigations as well as tandem affinity purification, which allow for the identification of interacting proteins, have also been included. The latter chapter highlights the beginning of our understanding of how genes (as words in a sentence) can be organized into a higher level of complexity so that words (genes and proteins) can be arranged into sentences (interacting genes/proteins). Methods for the study of immune response to fungal infections are highlighted in chapters on the evaluation of candidate vaccines, SIgA in protection, the interaction of fungi with dendritic cells, and phagocyte assays with fungi. Likewise, strain identification is vital in studies of pathogenesis and in clinical settings. This topic is discussed in chapters that provide these determinations by DNA fingerprinting or sensitivity to killer toxins. Finally, disease models of candidiaisis are described, and these discuss animal models as well as in vitro models (biofilm and tissue culture) that evaluate virulence. The text does not attempt to be inclusive for every current method, but rather the protocols most used are discussed. However, chapters reference alternative procedural approaches, and it is anticipated that the text will well serve the investigators as a source of methods in the field of medical and molecular mycology. Ronald L. Cihlar, PhD Richard A. Calderone, PhD v
- 12. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I IMMUNOGICAL METHODS 1 Isolation of Dendritic Cells from Human Blood for In Vitro Interaction Studies with Fungal Antigens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 T. Sreevalsan 2 Detection and Quantitation of Antifungal SIgA Antibodies in Body Fluids . . . . .9 Michael F. Cole 3 Phagocytosis and Killing Assays for Candida Species. . . . . . . . . . . . . . . . . . . . . .17 Chen Du and Richard A. Calderone 4 Immunization Protocols for Use in Animal Models of Candidiasis . . . . . . . . . . .27 Esther Segal and Hana Sandovsky-Losica PART II VIRULENCE AND BIOFILMS 5 Penetration of Antifungal Agents Through Candida Biofilms. . . . . . . . . . . . . . .37 L. Julia Douglas 6 Candida Biofilm Analysis in the Artificial Throat Using FISH . . . . . . . . . . . . . .45 Bastiaan P. Krom, Kevin Buijssen, Henk J. Busscher, and Henny C. van der, Mei 7 Conditions for Optimal Candida Biofilm Development in Microtiter Plates . . .55 Bastiaan P. Krom, Jesse B. Cohen, Gail McElhaney-Feser, Henk J. Busscher, Henny C. van der Mei, and Ronald L. Cihlar PART III VIRULENCE MEASUREMENTS : IN VITRO, EX VIVO, AND IN VIVO 8 Animal Models of Candidiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65 Cornelius J. Clancy, Shaoji Cheng and Minh Hong Nguyen 9 Candida albicans Gene Expression in an In Vivo Infection Model . . . . . . . . . . .77 Michael D. Kruppa 10 In Vitro and Ex Vivo Assays of Virulence in Candida albicans. . . . . . . . . . . . . . .85 Richard A. Calderone PART IV STRAIN TYPING AND IDENTIFICATION 11 Biotyping of Candida albicans and Other Fungi by Yeast Killer Toxins Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .97 Luciano Polonelli and Stefania Conti 12 DNA Fingerprinting Candida Species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117 Claude Pujol and David R. Soll vii
- 13. PART V GENOMICS AND PROTEOMICS 13 The Application of Tandem-Affinity Purification to Candida albicans . . . . . . .133 Chris Blackwell and Jeremy D. Brown 14 Preparation of Samples for Proteomic Analysis of the Candida albicans Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Neeraj Chauhan 15 Reporter Gene Assays in Candida albicans . . . . . . . . . . . . . . . . . . . . . . . . . . . .157 Joy Sturtevant 16 Genetic Transformation of Candida albicans . . . . . . . . . . . . . . . . . . . . . . . . . .169 Ana M. Ramon and William A. Fonzi 17 Large-Scale Gene Disruption Using the UAU1 Cassette. . . . . . . . . . . . . . . . . .175 Clarissa J. Nobile and Aaron P. Mitchell PART VI APPENDIX 18 Standard Growth Media and Common Techniques for Use with Candida albicans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197 Neeraj Chauhan and Michael D. Kruppa Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .203 viii Contents
- 14. Contributors CHRIS BLACKWELL Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom JEREMY D. BROWN Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom KEVIN BUIJSSEN Department of Biomedical Engineering and Departments of Otorhinolaryngology, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands HENK J. BUSSCHER Department of Biomedical Engineering, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands RICHARD A. CALDERONE Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA NEERAJ CHAUHAN Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA SHAOJI CHENG University of Florida College of Medicine and North Floirda/South Georgia Veterans Health System, Gainsville FL, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA RONALD L. CIHLAR Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA CORNELIUS J. CLANCY University of Florida College of Medicine and North Florida/ South Georgia Veterans Health System, Gainsville, FL, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA JESSE B. COHEN Department of Biology, Georgetown University Medical Center, Washington, DC, USA MICHAEL F. COLE Department of Microbiology and Immunology, Georgetown University Medical Center, NW, Washington, DC, USA STEFANIA CONTI Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Universitá degli Studi di Parma, Parma, Italy L. JULIA DOUGLAS Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glascow, Glascow, United Kingdom CHEN DU Department of Microbiology and Immunology, Georgetown University Medical Center, Washington DC, USA WILLIAM A. FONZI Department of Microbiology and Immunology, Georgetown University Medical Center, Washington DC, USA BASTIAAN P. KROM Department of Biomedical Engineering, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands MICHAEL D. KRUPPA Department of Microbiology, East Tennessee State University, James H. Quillen College of Medicine, Johnson City, TN, USA ix
- 15. GAIL MCELHANEY-FESER Department of Microbiology and Immunology, Georgetown University Medical Center, Washington DC, USA AARON P. MITCHELL Department of Microbiology Columbia University, New York, NY, USA MINH HONG NGUYEN University of Florida College of Medicine and North Florida/ South Georgia Veterans Health System, Gainsville FL, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA CLARISSA J. NOBILE Department of Microbiology Columbia University, New York, NY 10032, USA; Biological Sciences Program, Department of Biological Sciences, Columbia University, New York, NY, USA LUCIANO POLONELLI Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Universitá degli Studi di Parma, Parma, Italy CLAUDE PUJOL Department of Biological Sciences, The University of Iowa, Iowa City, IA, USA ANA M. RAMON Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, USA HANA SANDOVSKY-LOSICA Department of Human Microbiology Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel ESTHER SEGAL Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel DAVID R. SOLL Department of Biological Sciences, The University of Iowa, Iowa City, IA, USA T. SREEVALSAN Department of Microbiology and Immunology, Georgetown University Medical Center, Washington DC, USA JOY STURTEVANT Department of Microbiology, Immunology and Parasitology, Louisiana State University School of Medicine, New Orleans, LA, USA HENNY C. VAN DER MEI Department of Biomedical Engineering, University Medical Center Groningen and the University of Groningen, Groningen, The Netherlands x Contributors
- 17. Chapter 1 Isolation of Dendritic Cells from Human Blood for In Vitro Interaction Studies with Fungal Antigens T. Sreevalsan Abstract A method is described to generate dendritic cells (DCs) from human peripheral blood mononuclear cells (PBMCs). The procedure involves two major steps: (1) preparation of monocytes from human PBMCs and (2) in vitro differentiation of the monocytes into DCs by growth factors and cytokines. Cells obtained in this fashion are screened for the presence or absence of antigenic markers characteristic of DCs by flow cytometry. Key words: Dendritic cells, monocytes. 1. Introduction Dendritic cells (DCs) play a key role in the initiation of the primary immune response in a host following infection by a pathogen (1, 2). These cells are found in the body in two forms: an imma- ture and a mature form. Immature cells are present in every tissue and are efficient in processing naive antigens. Internalization and processing of an antigen by immature DCs lead to their migration to draining lymphoid organs where they undergo maturation. Mature DCs, though inefficient in antigen processing, can interact avidly with resting CD4 and CD8 T-cells resulting in their activa- tion. The maturation of DCs is accompanied by upregulation of CD80, CD86, adhesion molecules, cytokines, chemokines, and their receptors. These cells vary in their ability to transmit regulatory signals. DCs display considerable heterogeneity based on their developmental origin and interactions with various pathogens (3). Recently DCs have been used as a natural adjuvant in vaccination Ronald L. Cihlar, Richard A. Calderone (eds.), Candida albicans: Methods and Protocols, vol. 499 Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 DOI 10.1007/978-1-60327-151-6_1 Springerprotocols.com 3
- 18. and immunotherapy (4). Many studies using fungal cells have been reported indicating that DCs play an important role in the host defense against infections (5, 6). DCs act in vivo early during infection and mediate the uptake and the presentation of antigens to T-cells. These cells appear to connect the process of innate immunity with cell-mediated immunity in fungal pathogenesis (7). Many methods have been described in the literature to generate DCs on a large scale (8–10). In this regard, the technique reported by Danciger et al. (10) to prepare human DCs for studying in vitro interactions with fungal antigens has proven useful and is described below. The generation of DCs from human PBMCs by this techni- que involves two major steps: (1) isolation of pure monocytes from PBMCs and (2) differentiation of the monocytes into immature or mature DCs, which is described below. 2. Materials 2.1. Reagents 1. Heparin (Sigma), 2. Ficoll-Paque Plus and percoll (Amersham Biosciences), 3. Complete medium (CM): Iscove’s modified Dulbeccos’s medium (IMDM) (Invitrogen), 25 mM HEPES without phenol red (Life Technologies), 4. Fetal bovine serum (FBS) (Hyclone), 5. Dulbecco’s phosphate-buffered saline, 1 mM EDTA, pH 7.2 (DPBS), 6. Cytospin centrifuge (Shandon Elliot), 7. Protocol Hema 3 (Fisher). 8. The following reagents can be obtained from Becton Dickinson: anti-CD1a, anti-DCISGN, anti-CD14 and appropriate isotype sera, paraformaldehyde, and NaN3 and 9. FACS buffer: 2% FBS, 1% NaN3 in PBS lacking Ca++ or Mg++ . 3. Methods 3.1. Isolation of Monocytes 1. From healthy, fasting human volunteers, 360 mL blood is drawn into a container to which heparin (3.8 units/mL) has been added. 2. Aliquots of blood (30 mL) are removed to 50 mL polypropylene tubes (see Note 1). 4 Sreevalsan
- 19. 3. Then, 6 mL of DPBS is added to each tube. 4. The blood is then underlayered with 10 mL of Ficoll-Paque Plus. 5. Centrifuge at 400 g for 20 min at room temperature (RT). Leave the centrifuge break-off during this step. 6. A dense white band seen just above the red blood cells is removed carefully with a plastic 5 mL pipette. The banded cells are pooled in 15 mL aliquots. Fractions contain the PBMCs. 7. PBMC-containing aliquots (15 mL) are diluted with 35 mL of DPBS in siliconized centrifuge tubes (see Notes 2 and 3). The diluted cell suspension is centrifuged at 150 g for 10 min at RT. Platelets remain in the supernatant after this centrifugation. Each pellet is resuspended in 10 mL of DPBS and pooled. 8. The above step is repeated again to remove any remaining platelets. 9. Pellets (from four original aliquots) are pooled and suspended in 40 mL CM. Cell concentration is determined by counting in a hemacytometer. 10. The cells are diluted with CM (containing no phenol red) to a concentration of 1–2 106 in a sterile siliconized 500 mL flask. Aliquots of 25 mL are placed in 50 mL polypropylene centrifuge tubes. 11. The suspended cells are underlayered slowly and evenly with 25 mL of CM containing 46% iso-osmotic percoll (see Note 4). A 60 mL syringe and a long needle are used for the underlayering task. 12. The resulting discontinuous gradient is centrifuged at 550 g for 30 min at RT with the break off. After centrifugation, lymphocytes are found in the pellet, while the monocytes are found in the white band formed at the interface between the lower and upper portions of the gradient. 13. Monocytes are collected from the white band using a pipette. Bands from several such gradients are pooled. Aliquots of 15 mL from the pooled bands are diluted with 35 mL of ice-cold DPBS in 50 mL siliconized glass tubes that are on ice. 14. The cell suspension is centrifuged at 400 g for 10 min at 4°C. 15. Each pellet is resuspended in 20.5 mL of DPBS without EDTA. 16. An appropriately sized aliquot is used to determine cell density using a hemacytometer. Generation of Dendritic Cells from Human Blood 5
- 20. 3.2. Monocyte Differentiation into Dendritic Cells Differentiation of monocytes into immature DCs can be accom- plished by incubating them with the appropriate stimulating factor under study by the investigator. Likewise, immature or mature DCs are chosen to fit the investigation. For example, a combination of granulocyte-macrophage colony–stimulating factor (GM-CSF) and interleukin-4 (IL-4) produce immature DCs (11). Such immature DCs also express CD1a on their sur- face. After stimulation with other agents like lipopolysaccharide (LPS) or a combination of proinflammatory cytokines (IL-1beta, TNF-alpha, PGE2, and monocyte-conditioned medium (MCM)) (see Note 5), the immature DCs show increased expression of CD86, CD80, and MHC class II molecules, thus representing the mature DCs. 3.2.1. Immature DCs 1. The monocyte cell suspension prepared as described earlier is diluted with CM to a concentration of 6 106 /mL. 2. Cell suspension of 25 mL is placed in T-150 tissue culture flasks (Corning, NY) and incubated at 37°C for 2 h in order to allow cell adherence. 3. Gentle shaking of the flasks for a few minutes will allow the nonadherent cells to be released to the supernatant medium. 4. The medium is decanted. 5. The cell monolayer is washed with CM. 6. Fresh medium (35 mL) containing IL-4 at 1000 U/mL and GM-CSF (Schering-Plough) at 1000 U/mL are added and incubated at 37°C. 7. After 6 days incubation, the medium in the cultures is replenished with a fresh preparation of growth factors. 8. Two days later, the medium is decanted and nonadherent cells are removed from the cultures and saved. 9. The monolayers are washed gently with PBS to remove loosely adhered cells, leaving behind undifferentiated cells. 10. This fraction is combined with the first batch of nonadherent cells. 11. The cell suspension is centrifuged at 200 g for 10 min, washed with PBS as before, and resuspended in CM at the desired concentration. This represents the immature DCs. 3.2.2. Mature DCs 1. Steps 1–6 are identical to that described in Section 3.2.1. 2. After 8 days incubation, CM contains LPS or a cocktail of IL-beta, TNF-alpha, PGE2, and MCM. 3. The remainder of the procedure regarding handling the culture is identical to those described for obtaining immature DCs. 6 Sreevalsan
- 21. 3.3. Confirmatory Evidence for DCs Differentiation and Maturation Differentiation of monocytes into DCs is accompanied by upre- gulation of antigens CD1a and DCSIGN and a loss of CD14 expression. Thus, the DCs generated from monocytes should be screened for the above antigens using corresponding antibodies by flow cytometry. 1. Detached cells are suspended in 100 mL of FACS buffer. 2. Cells are incubated for 30 min on ice, following the addition of 5 mL conjugated anti-CD1a, anti-DCISGN, or anti-CD14, in the appropriate isotype serum (Becton Dickinson). 3. Samples are washed with FACS buffer. 4. Samples are fixed with 2% paraformaldehyde (Becton Dickinson) and stored at 4°C. 5. Samples are analyzed in a FACS flow cytometer (Becton Dickinson). 3.4. Continuous DC Cell Lines A few cell lines displaying the phenotypic and functional properties of DCs have been described in the literature (12–14). Upon differ- entiation, these cells seem to acquire some properties of DCs. Recently, such cell lines have been compared with primary DCs with respect to their ability to undergo both maturation and the accompanying functional changes (15). These cell lines were derived originally from human myeloid leukemia. The cell lines THP-1, KG-1, and MUTZ-3 were studied in this context. The results indicated that MUTZ-3 cells have comparable abilities in functional and transcriptional activity to those of the primary DCs. Therefore, MUTZ-3 cells can act as a model for primary DCs. The ease of cultivation of these cells in the laboratory makes them an ideal system to study the role of DCs in immune regulation. 4. Notes 1. Only sterile glassware or plasticware is used throughout the procedure. 2. Glassware can be siliconized by the following procedure: 50 mL glass centrifuge tubes (Kimax) are heated at 180°C for 4 h to inactive endotoxin, if any, and then cooled; the glassware is then coated with Sigmacote (Sigma). 3. Polypropylene tubes, Blue Max (Falcon) can be substituted for siliconized glassware. 4. Iso-osmotic percoll can be prepared as follows: CM with 46% percoll can be prepared by combining 46 mL of iso-osmotic percoll with 54 mL of CM. Phenol red is included in the CM to give contrast with the layer containing PBMC in medium Generation of Dendritic Cells from Human Blood 7
- 22. lacking phenol red. Iso-osmotic percoll is obtained by combin- ing 9.25 mL of percoll with 0.75 mL of 10X DPBS. 5. MCM can be prepared by incubating the monocyte culture (3–13) in CM for 24 h at a cell density of 2 106 cells/mL at 37°C. References 1. Stienman, R.M. (1991) The dendritic cell system and its role in immunogenicity. Ann. Rev. Immunol. 9, 271–296. 2. Hart, D.N. (1997) Dendritic cells: Unique leukocyte populations which control the pri- mary immune response. Blood 90, 3245–3287. 3. Shortman, K., and Liu, Y. (2002) Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2, 151–161. 4. Fong, L., and Engelman, E.G. (2000) Dendritic cells in cancer immunotherapy. Ann. Rev. Immunol. 18, 245–273. 5. Fe d’Ostiani, C., Del Sero, G., Bacci, A., Montagnoli, C., Spreca, A., Mencacci, A., Ricciardi-Casegnoli, P., and Romani, L. (2000) Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans: Implications for T helper cell immunity in vitro and in vivo. J. Exp. Med. 191, 1661–1674. 6. Bacci, A., Montagnoli, C., Perruccio, K., Bozza, S., Gaziano, R., Pitzurra, L., Velardi, A., Fe d’Ostiani, C., Cutler, J.E., and Romani, L. (2002) Dendritc cells pulsed with fungal RNA induce protective immunity to Can- dida albicans in hemapoietic transplanta- tion. J. Immunol. 168, 2904–2913. 7. Romani, L., Montagnoli, C., Bozza, S., Perruccio, K., Spreca, A., Allavena, P., Verbeek, S., Calderone, R.A., Bistoni, F., and Puccetti, P. (2004) The exploitation of distinct recognition receptors in dendritic cells determines the full range of host immune relationships with Candida albicans. Int. Immunol. 16, 149–161. 8. Romani, N., Reider, D., Heuer, M., Ebner, S., Kampgen, E., Eibl, B., Neiderwiesser, D., and Schuler, G. (1996) Generation of mature dendritic cells from human blood: An improved method with special regard to clin- ical applicability. J. Immunol. Methods 196, 137–151. 9. Tuyaerts, S., Noppe, S.M., Corthals, J., Breckpot, K., Heirman, C., Greef, C.D., Riet, I.V., and Thielemans, K. (2002) Generation of large numbers of dendritic cells in a closed system using cell factories. J. Immunol. Methods 264, 135–151. 10. Danciger, J.S., Lutz, M., Hamma, S., Cruz, D., Castrillo,A.,Lazaro,J.,Phillips,R.,Premack,B., and Berliner, J. (2004) Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemo- taxis, cellular adhesion assays, macrophage and dendritic cell differentiation. J. Immu- nol. Methods 288, 123–134. 11. Bender, A., Sapp, M., Schuler, G., Steinman, R.M., and Bhardwaj, N. (1996) Improved methods for the generation of dendritic cells from nonproliferating pro- genitors in human blood. J. Immumol. Methods 196, 121–135. 12. Koski, G.K., Schwartz, G.N., Weng, D.E., Czernieki, B.J., Carter, C., Gress, R.E., and Cohen, P.A. (1999) Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways. J. Immunol. 163, 82–92. 13. St. Louis, D.C., Woodcock, J.B., Franozo, G., Blair, P.J., Carlson, L.M., Murillo, M., Wells, M.R., Williams, A.J., Smoot, D.S., Kanshal, S., Grimes, J.L., Harlan, D.M., Chute, J.P., June, C.H., Soebelist, U., and Lee, K.P. (1999) Evidence for distinct intracellar signaling pathways in CD34+ progenitor to dendritic cells5 RRRRRR differentiation from a human cell model. J. Immunol. 162, 3237–3248. 14. Ackerman, A.L., and Cresswell, P. (2003) Regulation of MHC class I transport in human dendritic cells and dendritic–like cell line KG-1. J. Immunol. 170, 4178–4188. 15. Larsson, K., Lindstedt, M., and Borrebaek, C.A.K. (2006) Functional and transcrip- tional profiling of MUTZ-3, a myeloid cell line acting as a model for dendritic cells. Immunology 117, 156–166. 8 Sreevalsan
- 23. Chapter 2 Detection and Quantitation of Antifungal SIgA Antibodies in Body Fluids Michael F. Cole Abstract The measurement of antibodies in the external secretions that bathe mucosal surfaces is important in understanding the host response to the opportunistic pathogen, Candida albicans and its determinants of pathogenesis at these sites. The principal immunoglobulin isotype in mucosal secretions is secretory immunoglobulin A (SIgA). Unlike the circulatory system, mucosal surfaces are open systems in which the concentrations of immune factors are affected by diurnal variation, changes in flow rate, complex formation with mucins, and other variables. Thus, it is necessary to control these factors if meaningful data are to be obtained. This chapter outlines methods for the measurement of anti-Candida SIgA antibodies in primary units and shows how to control the factors that influence antibody measurement in external secretions. Key words: SIgA, SIgA antibodies, Candida antigens. 1. Introduction In studies of the pathogenesis of fungal infections or for the development of vaccines, it is frequently necessary to be able to detect and quantitate antibodies directed against the organism and/or its antigens in various body fluids. Body fluids can be divided into two types, those in closed systems such as blood, cerebrospinal fluid, peritoneal fluid, etc., and those in open systems, that is, mucosal secretions such as tears, nasal secre- tions, saliva, milk, genitourinary secretions, etc. While detection of antimicrobial antibodies in blood is generally quite straightforward, the detection and quantitation of antimicrobial antibodies in external secretionsisaltogetheradifferentproposition.Thereasonsforthisare several and include diurnal variation, the inverse relationship Ronald L. Cihlar, Richard A. Calderone (eds.), Candida albicans: Methods and Protocols, vol. 499 Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 DOI 10.1007/978-1-60327-151-6_2 Springerprotocols.com 9
- 24. between flow rate and antibody concentration, complexation of antibodies with high-molecular-weight mucins and other factors found in secretions, and proteolytic and gycolytic activity of the resident microbiotas that colonize mucosal surfaces. Detection of antibodies was revolutionized by the invention of the enzyme- linked immunosorbent assay (ELISA) by Engvall and Perlmann (1). This method is very versatile and sensitive, and is widely employed in both research and clinical care settings (2, 3). One limitation of the measurement of antimicrobial antibodies by ELISA is that the data are output in optical density units. While this may be satisfactory for making comparisons within individual laboratories,itmakescomparisonswithdatafromotherlaboratories difficult as optical density is affected by incubation time, source of antibody reagents, nature of the plastic plate, to name but a few. It is the purpose of this chapter to describe the application of ELISA to the detection of anti-Candida antibodies in external secretions in which the read-out is in primary units, rather than optical density. The method described combines the measurement of antimicrobial secretory IgA (SIgA) antibodiesand totalSIgAimmunoglobulin on a single 96-well-microtitration plate. The described method is applicable to the detection and measurement of almost all anti- microbial antibodies that are induced in the secretions of experi- mental animals or humans. The inexperienced reader is advised to familiarize themselves with the basic principles of solid-phase assays before embarking on the assay described in this chapter. A free technical handbook on ELISA and the related technique ELISPOT is available from Pierce Chemical at http://www.pierce net.com/Objects/View.cfm?Type=PageID=AF5B61C9-9149- 41F4-B63D-DA4C46CD9446 2. Materials 2.1. Collecting Mucosal Secretions 1. One piece 3.0 mL sterile disposable transfer pipette (Fisher Scientific, Pittsburgh, PA, USA). 2. Sterile phosphate-buffered saline (PBS), pH 7.4. 3. 500 mM ethylenediamine tetracetic acid (EDTA) solution. Dissolve 14.61 g of EDTA (Sigma-Aldrich, St. Louis, MO, USA) in 50 mL of deionized-distilled water (ddH2O) and filter sterilize using a 0.45 mm disposable vacuum filter unit. 2.2. Enzyme-Linked Immunosorbent Assay (ELISA) 1. 96-Well flat bottom microtiter plates (ImmuluxTM or Immu- luxTM HB, Dynex Technologies, Chantilly, VA, USA). 2. Coating buffer: 0.05 M carbonate buffer, pH 9.6, contain- ing 0.02% NaN3. Dissolve 1.6 g of Na2CO3 (anhydrous) 10 Cole
- 25. (MW ¼ 105.99), 2.9 g of NaHCO3 (MW ¼ 84.01), and 0.2 g of NaN3 (all from Sigma) in a final volume of 1 L of ddH2O. Store at 4°C. Discard after 2 weeks. 3. Blocking reagent: 0.1% (1.0 g/L) bovine serum albumin (BSA) fraction V (Sigma) containing 0.02% (0.2 g/L) NaN3 in PBS, pH 8.0. 4. PBS-Tween: 0.1% (1 mL/L) Tween 20 (polyoxyethylenesor- bitan monolaurate, Sigma) containing 0.02% (0.2 g/L) NaN3 in PBS, pH 8.0. 5. Horseradish peroxidase (HRP)-conjugated antibody diluent: 0.1% (1.0 g/L) BSA in PBS, pH 8.0. 6. Citrate–phosphate buffer: Mix four parts of 0.1 M citric acid (dissolve 1.92 g of anhydrous citric acid (Sigma) in 100 mL of ddH2O) with six parts of 0.2 M Na2HPO4 (dissolve 2.84 g of Na2HPO4 (Sigma) in 100 mL ddH2O). Adjust pH to 4.5, if necessary, with either solution. 7. HRP substrate solution: Dissolve 1 mg of o-phenylenediamine (Sigma) in 1.0 mL of citrate–phosphate buffer, pH 4.5 and add 0.012% H202 (4 mL of 30% H202 per 10 mL of buffer) (see Note 1). Prepare substrate solution immediately prior to use. Substrate is light sensitive; use dark bottle or wrap foil around the vessel. 3. Methods The method described below is designed to quantitate Candida- reactive SIgA antibodies in external secretions and output the data in primary units by interpolating optical density into a SIgA standard curve (ng/mL). In this application, Candida cells or purified Candida antigens form the solid phase, that is, they are immobilized on the plastic surface of the well of one-half of a 96-well-microtiter plate. Here they serve to capture SIgA antibo- dies reactive with them in the external secretion. On the other half of the plate, an antibody to human secretory component (SC) forms the solid phase and serves to capture a series of dilutions of purified SIgA, thus forming an SIgA standard curve. 3.1. Determination of the Dry Weight of Candida Cells 1. Dry a 25 mm, 0.4 mm Whatman Nuclepore1 polycarbonate filter (Fisher Scientific) to constant weight. Place the filter inside a P2O5 dessicator, evacuate the dessicator and place it overnight in a 60°C incubator. 2. Weigh individual filters on an analytical balance accurate to 10 mg, and record weight on Petri dish. Antifungal SIgA Antibody Detection 11
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- 27. 1240 106 At centre-way between maincap and topmast-crosstrees; 4 feet over foretopsail- yard (hoisted); cap of mizentopmast. 1299 130 At maintopmast cap; 7 feet under head of foretopgallant rigging. With 2 shot the elevation must be nearly double that which, with 1 shot, and the same charge of powder, produces the same range. The angles of elevation, corresponding to the ranges, increase, by quarter degrees, from point blank. *The reason for transferring the sight to the line-of-metal is, obviously, to use the dispart elevation for the purpose of getting a more direct view. Vide Tables of Practice, c., pages 74, 75.
- 28. TABLE E. Tangent practice with short 24, and 18-Prs., with 1 solid shot and a charge of ¼th the shot’s weight, from the maindeck of a 2nd class Frigate; the height of the Gun above the surface of the water being 7 feet 6 inches. Distance in yards. Take aim. Height of parts aimed at. Point at the undermentioned parts of Frigates of 44 guns. 221 P. B. . . Point at part intended to hit. By sight parallel to axis of bore. 312 11 ft. 6 in. At 2 feet below the level of the quarterdeck, gangway, and forecastle. 403 18 6 At bulwark rail of quarterdeck, gangway, and forecastle. 494 26 6 At 6 feet over the upper part of hammocks stowed in quarterdeck, gangway, c. 582 37 6 At 2 feet under centre of mainmast, reckoning from top of hammocks to mainyard: 2 feet over corresponding mark in foremast. 644 50 At 8 feet under mainyard; 1 foot under foreyard; crossjack-yard. 706 62 At 2 feet under half-way from mainyard to maintop; 1 foot under foretop; mizentop. 768 78 At half-way from maintop to maincap; 2 feet over forecap; 7 feet over mizencap. 832 94 At 10 feet over maincap or one-third up to the topmast, reckoning from cap to topsail-yard, (hoisted); 6 feet under foretopsail-yard, hoisted; mizentopsail-yard. 907 .. . . Point at part intended to hit. *By the line of metal. 982 20 6 At upper part of the hammocks stowed in quarterdeck, forecastle, nettings, c.
- 29. 1057 35 6 At 4 feet under centre of mainmast, reckoning from top of hammocks to mainyard; centre of foremast. 1133 51 At 7 feet under mainyard; foreyard; crossjack-yard. 1177 69 At 1 foot under maintop; centre between foretop and forecap; 2 feet under mizencap. 1221 87 At 3 feet over maincap; 1 foot under centre between maincap and topsail- yards (hoisted up); 4 feet under centre of mizentop-sail. 1265 106 At 8 feet under maintopsail-yard (hoisted); 3 feet under foretopmast crosstrees; 1 foot over mizentopmast cap. 1308 127 At 1 foot under maintopmast cap; 9 feet under the head of foretopgallant- rigging; 7 feet over the head of mizentopgallant-rigging. With 2 shot the elevation must be nearly double that which, with 1 shot, and the same charge of powder, produces the same range. The angles of elevation, corresponding to the ranges, increase, by quarter degrees, from point blank. *The reason for transferring the sight to the line-of-metal is, obviously, to use the dispart elevation for getting a more direct view. Vide Tables of Practice, c., pages 74, 75.
- 30. INSTRUCTIONS FOR THE EXERCISE, AND SERVICE OF GREAT GUNS, AND SHELLS ON BOARD HER MAJESTY’S SHIPS. Positions. (Vide Plate.) Before loading. Loading. Training. Gun Numbers. 1, 2, 3, 4, 5, 6. Auxiliaries. 7, 8, 9, 10, 11, 12, 13, c. Handspike-men. 9, 10. Rear-men. 12, The right rear-man. 13, The left rear-man, or the two highest numbers. Words of command. “Prime.”—“Point.”—“Elevate.”—“Ready.”—“Fire.”—“Stop the vent.”—“Sponge.”—“Load.”—“Run out.” Number of Men allowed to the following Broadside guns. Nature of Gun. Weight. Length. Number of men. Carriage. cwt. Feet. 8-inch. 65 9 14 } { 56 9½ 13 } These guns are mounted on common carriages. { 50 9 12 } 32 Pounder. { 45 8½ 11 } { 42 8 10 } { 32 6½ 9 } Mounted on Hardy’s carriages. { 25 6 8 }
- 31. 32 Pr. Carronade 17 4 7 } POSITIONS. J. W. Lowry, sc. Before Loading. Loading. Training. Stationary Powderman Stationary Powderman Stationary Powderman Extra Powderman Exercise with 13 Men, to a Lower deck gun. No. 1. The Captain;—commands, attends the breeching, bed, and quoin, primes, points, fires, and stops the vent. 2. The second Captain;—assists No. 1, casts loose, hauls up the port, runs out, attends the apron, and port tackle-fall, and cocks the lock.
- 32. 3. Takes out the tompeon, bears out the port, loads, rams home, runs out, and trains. 4. Takes out the tompeon, bears out the port, worms, sponges, rams home, runs out, and trains. 5. Casts loose, hauls up the port, gives shot and wad to No. 3, runs out, trains, and spans the breeching. 7, and 8. Cast loose, run out, and train. 9, and 10. Cast loose, run out, and attend handspikes. 11. Casts loose, runs out, and trains. 12. Casts loose, and attends train tackle. 13. Casts loose, runs out, trains, and fires with a hammer, or match. Note.—The duties of No. 2 for upper, or main deck guns will be the same as that for lower deck guns, omitting the words “haul up the port,” and “attends port tackle-fall.” The duties likewise of Nos. 3, and 4 will be the same, substituting the words “takes off the upper half- port,” “lets down the lower one,” or “takes out the port” (as the case may be) for “bears out the port.” The duties also of Nos. 5, and 6 will be the same, omitting the words “hauls up the port.” Handspike-men with 5, 6, or 7 men Nos. 5, and 6 ” ” 8 or 9 men ” 7, and 8 ” ” 10 or 11 men ” 7, and 8 and assistant handspike-men ” 9, and 2 Handspike-men with all Nos. above 11 ” 9, and 10 and assistant handspike-men ” 11, and 2 With light guns it may be advantageous, in some cases, to double man the handspikes. The left rear-man will always fire with a hammer, or match; and the right rear-man will attend the train tackle, except when he is handspikeman (when No. 2 will attend it), and in lower deck exercise (when the left rear-man will attend it).
- 33. At the word “Man both sides,” each watch will repair to its respective side, the odd numbers standing to the left of the left guns; even numbers to the right of the right guns. “Man both sides.” Left guns.—No. 3 remains 3; 5 becomes 4; 7—6; 9—5; 11—2; 13— 7; 1 remains 1. Right guns.—No. 4 remains 4; 6 becomes 3; 8—6; 10—5; 12—2; 2 —1. Note.—The left guns are odd starboard, and even port. The right guns are even starboard, and odd port. The odd numbered guns’ crews are taken from the starboard watch; the even numbered from the port watch. Guns’ crews always man, and powder boys always supply adjacent guns, when clearing for action, or when fighting both sides. Note.—With a crew of 11 men, and upwards, and both sides manned, No. 2 is always to attend the train tackle. When casualties occur at the guns, those holding the highest numbers, or those last placed, will be the first to move to fill up the vacancies, excepting that where both captains are removed, the officer will name the most fitting person to become No. 1, filling up the vacancy as above. For instance; if there should be 13 men at the gun, and Nos. 3, 6, and 9, are ordered to “fall out,” Nos. 5, and 7, move up, becoming Nos. 3, and 5; No. 11, moves up, and becomes No. 7; No. 13,—No. 9; No. 8 moves up, and becomes No. 6; No. 10, —No. 8; and No. 12,—No. 10.[32] Exercise with 7 Men, to a 32-pounder carronade. No. 1. The Captain; commands, attends the breeching, primes, points, fires, and stops the vent. 2. The second Captain; assists No. 1, casts loose, runs out, attends the apron, and elevating screw, cocks the lock, and attends
- 34. train-tackle. 3. Takes out the tompeon, takes off the upper half-port, lets down the lower one, loads, rams home, runs out, and trains. 4. Takes out the tompeon, takes off the upper half-port, lets down the lower one, worms, sponges, rams home, runs out, trains, and attends compressor, when the gun is out. 5. Casts loose, gives shot, and wad to No. 3, runs out, trains, and spans the breeching. 6. Casts loose, gives sponge, rammer, and worm to No. 4, runs out, trains, spans the breeching, and attends compressor, when the gun is in. 7. Casts loose, runs out, trains, and fires with a hammer, or match. Note.—Handspikes are used with all guns, and sometimes with carronades. The duties with 9 men at the 32 cwt., and 8 men at the 25 cwt. guns are the same as with the 32-pounder carronade, except that No. 8 attends the compressor, instead of No. 6. Arrangement for Fighting both sides. When ordered to quarters, each watch will take its respective side; when the crew will assume the Nos. to which the several duties, prescribed for working the guns, are assigned. In the event of being attacked on both sides, at the same time, the following distribution is to be made, where 6 men, and upwards, besides the powderman, can be allowed to each gun, and its opposite, viz.:— Fighting both sides. With 6 men, and powderman. With 8 men, and powderman. With 10 men, and powderman. With 12 men, and powderman. 3 ┃ 4 3 ┃ 4 3 ┃ 4 3 ┃ 4 3 ┃ 4 3 ┃ 4 3 ┃ 4 3 ┃ 4
- 35. ┃ ┃ ┃ ┃ ┃ ┃ ┃ ┃ ‖┃‖ ‖┃‖ ‖┃‖ 6 ‖┃‖ 6 5 ‖┃‖ 6 5 ‖┃‖ 6 5 ‖┃‖ 6 5 ‖┃‖ 6 ┃ ┃ ┃ ┃ ┃ ┃ ┃ ┃ ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ 2 ‖┃‖ 2 1 1 1 1 1 1 1 1 Powder- man. Powder- man. Powder- man. Powder- man. Powder- man. Powder- man. Powder- man. Powder- man. 1 1 1 1 1 1 1 1 ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ ‖┃‖ 2 ‖┃‖ 2 ‖┃‖ 2 ‖┃‖ 7 ┃ ┃ ┃ ┃ ┃ ┃ ┃ ┃ ‖┃‖ 6 ‖┃‖ 6 ‖┃‖ 6 ‖┃‖ 5 6 ‖┃‖ 5 6 ‖┃‖ 5 6 ‖┃‖ 5 6 ‖┃‖ 5 ┃ ┃ ┃ ┃ ┃ ┃ ┃ ┃ 4 ┃ 3 4 ┃ 3 4 ┃ 3 4 ┃ 3 4 ┃ 3 4 ┃ 3 4 ┃ 3 4 ┃ 3 With 7 men, and powderman. With 9 men, and powderman. With 11 men, and powderman. With 13 men, and powderman. The guns are to be worked in pairs, commencing from forward; Nos. 1, and 2 guns on each side will be pairs, and so on, up to the highest even numbered gun on the deck; but the aftermost (if it should be an odd numbered gun) must be worked by its own crew passing from side to side, as necessary. When exercising, until the crews are perfect, and steady, the orders should be given by the officer, who is in charge of the quarters, and the left guns should be fired first. But when the men are perfect, the guns, which are first ready, should be first fired, and the exercise should be conducted by the captains of the guns, having reference to the movements of the other gun of their pairs, in order to approximate the exercise, as nearly as possible, to action with an enemy. All shot practice at targets, with both sides manned, is to be conducted on this plan. Words of command for Fighting both sides.
- 36. “Man both sides.”—“Prime.”—“Point.”—“Muzzle to the right.”—“Muzzle to the left.”—“Elevate.”—“Ready.”—(Left guns.) —“Fire.”—“Sponge, and load.”—(Right guns.)—“Fire.”—“Sponge, and load.”—(Left guns.)—“Run out.”—“Fire.”—“Sponge, and load.”— (Right guns.)—“Run out.”—“Fire.”—“Cease firing.” When ordered to “cease firing,” the guns are to be loaded, and run out.[33] Exercise for the 10-inch, or other revolving Gun, with a crew of 17 men. The crew are assembled as in the established Gun exercise; then — Gun.—Nos. 1, 2, 3, 4, 5, 6. Auxiliaries.—Nos. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. Traversing tackle-men.—Nos. 7, 8. Handspike-men.—Nos. 9, 10. Assistant handspike-men.— Nos. 11, 12. Compressor men, Nos. 13, 14. Rear-men.—No. 16, the right rear-man. No. 17, the left rear- man. No. 1. The Captain. No. 4. Sponger. ” 2. Second Captain. ” 5. Assistant loader. ” 3. Loader. ” 6. Assistant sponger. Words of command. “Traverse.”[34] —“Prime.”—“Point.”—“Elevate.”—“Ready.”—“Fire.”— “Stop the vent.”—“Sponge.”—“Load.”—“Run out.” Exercise with 17 men. No.
- 37. 1. The Captain; commands, attends the breeching, bed, and quoin, primes, points, fires, and stops the vent. 2. Second Captain; assists No. 1, casts loose, runs out, attends the apron, cocks the lock, and attends rear bolt. 3. Takes out the tompeon, loads, rams home, runs out, attends fighting bolt, shackles, and unshackles breeching, if necessary. 4. Takes out the tompeon, worms, sponges, rams home, runs out, attends stop-handspike, shackles, and unshackles breeching, if necessary. 5. Casts loose, runs out, traverses, and spans the breeching. 6. Casts loose, gives sponge, rammer, and worm to No. 4, runs out, traverses, and spans the breeching. 7, and 8. Cast loose, run out, attend traversing tackles, and shift side tackles. 9, 10, 11, and 12. Cast loose, run out, and attend handspikes. 13, and 14. Cast loose, run out, traverse, and attend compressors. 15. Casts loose, runs out, and traverses. 16. Casts loose, traverses, shifts traversing-tackle, brings up shot, or shell, attends stop-handspike, and train-tackle. 17. Casts loose, traverses, shifts traversing-tackle, brings up shot, or shell, fires with a hammer, or match, and attends train tackle. N.B. These numbers will be reduced for lighter guns, as may be necessary, when Nos. 11, and 12, will attend compressors, and the rear-men will do the duties of No. 16, and 17. When slide-guns are fitted with Ferguson’s Compressor, No. 6 is to attend to it. On coming to the gun, one, or other of these orders will follow; viz.,—“Action on the rear bolt,” or “Action on the fighting bolt required.” No. 2, attends the rear, No. 3, the fighting bolt, No. 1 gives the word “Right, or left traverse;” and when bearing on the object, or
- 38. when on the fighting bolt required “Well:” the gun is then to be wormed, sponged, loaded, and run out, without further orders. MORTAR EXERCISE. Mortars— 13-inch Land service— Nos. 1, 2, 3, 4, 5, 6. ” 10-inch ” ” ” 1, - 3, 4, 5, 6. ” 8-inch ” ” ” 1, - 3, 4, - 6. Words of command. “Point.”—“Run the mortar up.”—“Cross lift the mortar to the right.”—“Cross lift the mortar to the left.”—“Muzzle to the right.”—“Muzzle to the left.”—“Down.”—“Load.”—“Prime.”—“Fire.” 13-inch Land service Mortar. Duties.—No. 1. Commands, points, and serves the vent. 2. Serves ammunition. 3. Loads, assists to put in the shell, runs up, and trains. 4. Sponges, wipes the bottom of the shell, uncaps the fuse, puts in shell, runs up, trains, and attends sheepskin. 5. Assists to bring up shell, runs up, trains, and fires. 6. Brings up shell, guides it into the mortar, runs up, trains, and primes. 10-inch Land service Mortar. Duties.—No. 1. Commands, points, and serves the vent. 3. Serves ammunition, loads, assists to put in shell, runs up, and trains. 4. Sponges, wipes the bottom of the shell, uncaps the fuze, puts in shell, runs up, trains, and attends sheepskin. 5. Assists to bring up shell, runs up, trains, and fires.
- 39. 6. Brings up shell, runs up, trains, and primes. 8-inch Land service Mortar. Duties.—No. 1. Commands, primes, points, and serves the vent. 3. Serves ammunition, loads, runs up, and trains. 4. Sponges, wipes the bottom of the shell, uncaps the fuze, puts in shell, runs up, trains, and attends sheepskin. 6. Brings up shell, and fires. To dismount a 13-inch Land service Mortar, with any number of men, not less than 14. Duties.—No. 1. Commands. 2. Assists No. 1, and lashes handspikes. 3, and 4. Unlash the quoin, and place handspikes in the mortar. 5, and 6. Bring up a drag-rope each, and place the loop ends over the handspikes. All the Nos. man the dragropes, except Nos. 3, and 4, who attend with their handspikes, and raise the mortar perpendicular; then Nos. 3, and 4 steady the mortar, place the dragropes round the body of the mortar, and take out the handspikes and quoin; Nos. 5, 6, 7, and 8 unscrew, and take out the cap-squares. All the numbers man the dragropes, except Nos. 3 and 4, and throw the mortar to the rear. If it does not fall on its face, Nos. 3, and 4 place their handspikes under the trunnions (Nos. 5, and 6 the bight of the dragropes under the trunnions) and raise the mortar on its face; then Nos. 3, and 4 place their handspikes on the trunnions; Nos. 5, and 6 place the loop ends round the handspikes, and trunnions; even Nos. go to the front, and odd Nos. to the rear, and turn the mortar round. Nos. 5, and 6 place their dragropes over the opposite bolts of the mortar bed; Nos. 3, and 4 assist with their handspikes. All the Nos., at right angles to each other, turn the mortar bed round, and run it close up to the mortar.
- 40. To mount the Mortar. Nos. 3, and 4 place their handspikes under the trunnions, and on the top steps of the mortar bed; Nos. 5, and 6 place their dragropes round the trunnions, and throw the mortar into the trunnion boxes: the mortar is raised perpendicular, as before. All the Nos. will do what they have undone; even Nos. go to the front, odd Nos. to the rear, and ease the mortar down. N.B. The cap-squares are not to be shifted over till the mortar is raised perpendicular. INSTRUCTIONS FOR LANDING SEAMEN, AND MARINES, WITH FIELD PIECES. For Exercise, or Service on shore. 1. The boats should be formed in divisions, according to the seniority of the Captains of their respective ships, numbering from No. 1, on the right. The seamen, and marines should be told off in companies previous to leaving their ships, and on landing they will form immediately in the same order. 2. Each division of boats should have a distinguishing flag. Launches will carry two scaling ladders, intrenching tools, c.; barges, and pinnaces, one ladder each. 3. The boats will always land a boat’s length apart. Before leaving the ships, two boat keepers for each boat, and an officer in charge of each division of boats are to be told off, and are on no account to leave them. A fast-pulling boat with medical officers will attend in rear of the line. 4. Should the distance from the point of landing be considerable, the boats of each division, in tow of each other, commencing with the lightest boats, will pull in,—the leading boat of each division abreast, leaving space for the whole to form line abreast when ordered.
- 41. The boats will be dressed in line, as well as circumstances will admit. The officer in command will commence firing from the gun- boats when he thinks fit, but no shells, Shrapnell shells, or musketry are to be fired without orders. 5. When the commanding officer perceives the beach to be cleared (or when he considers it proper) he will order the boats to advance; they will pull in immediately, and land their crews, and field pieces, the latter will be formed on the flanks of their own division, or in batteries, according to orders; the scaling ladders in the rear until required for service. 6. The launches’ crews may be employed in throwing up a breast- work, and placing their guns in it, to cover their re-embarkation, should it become necessary. 7. Should the boats be employed for the disembarkation of troops, the same arrangement as to the divisions of boats should remain. It will then be desirable that every boat should carry a flag similar to that of the commanding officer of its division, and, when in large numbers, the boats should also be painted according to the colours of the flags, that the troops may readily know their own boats. On these occasions, the launches, barges, and pinnaces will form a front line, so as to clear the beach; the light boats will tow troop boats, paddle box boats, c., and be ready to succour any boats that may be damaged by the enemy’s fire. The orders, as to the firing of musketry, should be strictly enjoined, as before, with the seamen, and marines. It would be better that no musket should be loaded, and no knapsacks taken. Proportion of Charges, Spare powder, c., for a 51 Gun, screw, steam Frigate; and 50 Gun Frigate. KEY: A = Number. B = Proportion for each Gun. C = Total number of Cartridges.
- 42. D = Number in each case. E = Number of cases. 51 Gun, screw, steam Frigate. Nature of Ordnance. A Charge. B C D E Spare powder. Remarks. 68 Pr. 95 cwt. 1 Distant 16 lb. Full 10 lb. 120 120 6 20 20 rounds per gun, full charges, as spare; amounting to 200 lb. This spare powder would amount to 5560 pounds, and would be packed in 15 pound bags, 8 in a case, and would stow in 47 cases. 60 60 11 7 8 in. Gun 65 cwt. 12 Reduced 5 lb. Full 8 lb. Distant 10 lb. 20 240 15 16 20 rounds per gun, reduced charges, as spare; amounting to 1200 lb. 40 480 14 34½ 20 240 11 22 32 Pr. 58 cwt. 18 Reduced 6 lb. Full 8 lb. Distant 10 lb. 40 720 19 38 20 rounds per gun, reduced charges, as spare; amounting to 2160 lb. 20 360 14 26 20 360 11 33 32 Pr. 45 cwt. 20 Reduced 5 lb. Full 7 lb. 30 600 22 27½ 20 rounds per gun, reduced charges, as spare; amounting to 2000 lb. 50 1000 16 62½ 24 Pr. Howitzer 12½ cwt. 2 2½ lb. 174 348 20* 17† 2 half-cases of bursters, (10 oz., 6 oz., 5½ oz., and 4½ oz.,) and two half-cases of fuses. 12 Pr. Howitzer 6 cwt. 2 1¼ lb. 174 348 40* 9‡ * In a half-case. † Half-cases, and a quarter-case. ‡ Half-cases. KEY: A = Number. B = Proportion for each Gun. C = Total number of Cartridges.
- 43. D = Number in each case. E = Number of cases. 50 Gun Frigate. Nature of Ordnance. A Charge. B C D E Spare powder. 8 in. Gun 65 cwt. 8 Reduced 5 lb. Full 8 lb. Distant 10 lb. 20 160 15 11 20 rounds per gun, reduced charges, as spare; amounting to 800 lb. 40 320 14 23 20 160 11 15 32 Pr. 56 cwt. 22 Reduced 6 lb. Full 8 lb. Distant 10 lb. 40 880 19 46 20 rounds per gun, reduced charges, as spare; amounting to 2640 lb. 20 440 14 32 20 440 11 40 32 Pr. 45 cwt. 20 Reduced 5 lb. Full 7 lb. 30 600 22 27½ 20 rounds per gun, reduced charges, as spare; amounting to 2000 lb. 50 1000 16 62½ 24 Pr. Howitzer 12½ cwt. 2 2½ lb. 174 348 20* 17† 2 half-cases of bursters, (10 oz., 6 oz., 5½ oz., and 4½ oz.,) and two half- cases of fuses. 12 Pr. Howitzer 6 cwt. 2 1¼ lb. 174 348 40* 9‡ * In a half-case. † Half-cases, and a quarter-case. ‡ Half- cases. ON NAVAL BOMBARDMENTS.[35] “The attack of fortresses, and powerful land batteries with a naval force only, must ever be a hazardous, and perhaps desperate undertaking. But if skilfully combined with a military force sufficiently strong to make good its landing, to invest the place, or the batteries
- 44. on the land side, to take the defences in reverse, and so open the way to the attack by sea, the object of the attack will in general be successful. But this mode of proceeding can only be applied when the place to be attacked occupies a position, insular or otherwise, of such extent as to admit of being attacked by land as well as by sea. When the place, fortress, or arsenal to be attacked is covered and protected by isolated points of defence, mutually protecting each other, and when no previous military operation can be made, those points or outposts should be attacked in detail, and successively reduced; after which the fleet may arrive at, and attack the main position. This must evidently be a protracted and difficult process, even with such means: with ships alone, it cannot be effected without severe loss, and damages: and it should always be remembered that many of the attacking ships would be severely injured, probably disabled, in the attempt, whilst the enemy’s fleet would remain untouched, and in reserve. It would, therefore, follow that the attacking fleet must be exposed to a very disadvantageous action with the enemy, in the event of the latter subsequently leaving his place of shelter. “When the fortress, or arsenal to be attacked is situated on a coast which may be approached from the open sea in any direction, steam-ships may avoid the danger of a direct attack, end-on, or oblique, by approaching the place on either, or perhaps on both sides; and, having gained the proper proximity, clear of raking, or diagonal fire, range quickly up in parallel order, to attack the place in line, or lines; as in steam warfare, ship against ship, or fleet against fleet, direct advances upon the broadside batteries of ships, may, upon the same principle be avoided, and the enemy attacked in parallel order, by ranging up to him, and forced to fight if the attacking ships are superior in speed. “But when the fortress, arsenal, or place to be attacked is only approachable by a narrow and intricate channel, through which ships can only pass singly, or nearly so, there can be no manœuvering for position. There is no way of avoiding being met by direct, then oblique, and ultimately raking fire from the batteries that defend the channel, and steam can only perform its office of propulsion into or
- 45. through those intricacies under these disadvantageous, and hazardous circumstances. Steam-ships might, indeed, run past advanced, or covering batteries at full speed, without being much damaged; but it would be extremely perilous to leave such forts unsilenced in their rear, and, unless the daring enterprise should succeed, like Nelson’s, at Copenhagen, to produce a cessation of hostilities, the fleet, or at least any disabled ships, could never get out again. “However successful a naval attack of a fortress, or arsenal may be, the work of destruction can never be effectually accomplished by ships. The sea defences may be silenced, guns dismounted, parapets ruined, magazines blown up by mortar shells, and habitations devastated by the cruel process of bombardment; but no substantial demolition of the defences, or material destruction of public works and property, can be effected, unless the partial and rather temporary than permanent damages inflicted by the attacks of ships be followed up and completed, by having actual possession of the captured place for a sufficient time to ruin it entirely. No naval operation, however skilfully planned, and gallantly executed, can, in this way, reap the fruits of its own victory. “In the desultory operations of small active steamers employed to shell, with their pivot guns, open towns, roadsteads, harbours, and slender buildings, magazines, stores, c., c., or to shell bodies of troops on shore, the attacking vessels should never anchor, but having given their end-on fire, go off at speed to reload, and prepare to take up the fire in turn with others, whenever they regain a favourable position for a good effect. To hit a steamer running with speed across a line of fire is no easy matter (Arts. 331, 341); and when in the end-on position, she presents but a small target to hit at a long range.”
- 46. PA R T I X . BATTERIES, AND FORTIFICATION. BATTERIES. A battery, in respect to its profile, may be either elevated, half sunken, or sunken; and it is usually reveted with gabions, fascines, sand-bags, c. An elevated battery has its whole parapet raised above the natural surface of the ground, and, to procure the mass of earth required, a ditch is usually dug directly in front of the parapet. A half-sunken battery has its interior space, or terreplein, sunk some inches below the natural surface, and its parapet is composed of the earth thus obtained, and of that taken from a narrow ditch in front. A sunken battery has the whole of the earth taken from the interior space to form the parapet; and it must therefore be lowered from 2 feet to 3 feet 6 inches, according to the height of the gun carriages to be used. The half-sunken battery is constructed the quickest, as the diggers can work both in front and rear, at the same time. In a sunken battery, the diggers are as much crowded as in an elevated one, but, since the mass of parapet to be raised is smaller, it may be completed in much less time. Casemates, or vaulted batteries, are made bomb-proof, and the embrazures are cut through the revetment. Barbet batteries have no embrazures, the guns being placed on traversing platforms to enable them to fire over the parapet.
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