![the unmoved commander. At last, just as they got abreast of the
foremost junks, between which he had steered, the words
"Commence firing" rang out from Woodward's lips, and at the same
instant he signalled "Stop her" to the engineers.
The men sprang up with a cheer of defiance, and poured a
discharge of grape and canister into the junks on either side, (flash)
bang (flash—flash—flash) bang—bang—bang—(flash) bang; and the
excited sailors loaded and fired with tremendous energy. In a very
short time a thick pall of smoke completely enveloped the ship, and
with great difficulty the captain managed to keep her in position
between the line of junks—she in the mean time drifting slowly
ahead. After the first few discharges the men lost their hearing
through the stunning reports, and would vainly bawl at each other,
while their bodies were grimed with the smoke of the powder, every
one of them being stripped to the waist. The powder-monkeys were
as active as their namesakes, feeling their way in the thick smoke,
so as to avoid being knocked down by the rammers or sponges, and
cautiously treading clear of the tackle laid along the decks. It was
wonderful how clever the youngsters were, and with what accuracy
they would return to their own guns, although it was impossible to
see them. The flashes, which at first dazzled their eyes, now merely
made them blink for a moment, while their dulled ears only heard a
faint boom, and after a time did not notice even that.
Woodward sprang up aloft, and saw the ship was heading right, and
that the first two junks which they had passed were on fire. Upon
his return to the deck he met the master, who bawled something in
his ear; but as he could not understand what he said,[1] he
motioned him to go aloft, and keep a look-out.
Although the Stinger steamed quite slowly between the lines of
junks, she had not lost a man; and the pirates being unable to
depress their guns sufficiently to hit the ship very often, had actually
been firing into each other. When Woodward found that the shots
were striking the ship in an oblique direction he rang the signal, "Go](https://image.slidesharecdn.com/9654-250504071724-4d8333b8/85/Transgenesis-Techniques-Principles-and-Protocols-3rd-Edition-Leonie-Ringrose-Auth-41-320.jpg)
Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.)
- 1. Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.) download https://ebookultra.com/download/transgenesis-techniques- principles-and-protocols-3rd-edition-leonie-ringrose-auth/ Explore and download more ebooks or textbooks at ebookultra.com
- 2. We have selected some products that you may be interested in Click the link to download now or visit ebookultra.com for more options!. Aerosol Measurement Principles Techniques and Applications 3rd Edition Pramod Kulkarni https://ebookultra.com/download/aerosol-measurement-principles- techniques-and-applications-3rd-edition-pramod-kulkarni/ Nuclear Transfer Protocols Cell Reprogramming and Transgenesis 1st Edition Marie A. Di Berardino (Auth.) https://ebookultra.com/download/nuclear-transfer-protocols-cell- reprogramming-and-transgenesis-1st-edition-marie-a-di-berardino-auth/ Bioseparations Principles and Techniques B. Sivasankar https://ebookultra.com/download/bioseparations-principles-and- techniques-b-sivasankar/ Germ Cell Protocols Vol 2 Molecular Embryo Analysis Live Imaging Transgenesis and Cloning Methods in Molecular Biology Vol 254 Heide Schatten https://ebookultra.com/download/germ-cell-protocols-vol-2-molecular- embryo-analysis-live-imaging-transgenesis-and-cloning-methods-in- molecular-biology-vol-254-heide-schatten/
- 3. Satellite Networking Principles and Protocols 1st Edition Zhili Sun https://ebookultra.com/download/satellite-networking-principles-and- protocols-1st-edition-zhili-sun/ Satellite Networking Principles and Protocols 2nd Edition Zhili Sun https://ebookultra.com/download/satellite-networking-principles-and- protocols-2nd-edition-zhili-sun/ Microfluidic Techniques Reviews and Protocols 1st Edition Shelley D. Minteer https://ebookultra.com/download/microfluidic-techniques-reviews-and- protocols-1st-edition-shelley-d-minteer/ Plant pathology techniques and protocols 1st Edition Isla A. Browning (Auth.) https://ebookultra.com/download/plant-pathology-techniques-and- protocols-1st-edition-isla-a-browning-auth/ Bio Imaging Principles Techniques and Applications 1st Edition Rajagopal Vadivambal https://ebookultra.com/download/bio-imaging-principles-techniques-and- applications-1st-edition-rajagopal-vadivambal/
- 5. Transgenesis Techniques Principles and Protocols 3rd Edition Leonie Ringrose (Auth.) Digital Instant Download Author(s): Leonie Ringrose (auth.), Elizabeth J. Cartwright (eds.) ISBN(s): 9781603270199, 1603270191 Edition: 3 File Details: PDF, 4.07 MB Year: 2009 Language: english
- 6. ME T H O D S I N MO L E C U L A R BI O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651
- 7. i i i Transgenesis Techniques Principles and Protocols Third Edition Edited by Elizabeth J. Cartwright CardiovascularMedicine,UniversityofManchester,Manchester,UK
- 8. iv ISSN: 1064-3745 e-ISSN: 1940-6029 ISBN: 978-1-60327-018-2 e-ISBN: 978-1-60327-019-9 DOI: 10.1007/978-1-60327-019-9 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009929336 © Humana Press, a part of Springer Science+Business 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 is part of Springer Science+Business Media (www.springer.com) Editor Elizabeth J. Cartwright Cardiovascular Medicine University of Manchester Manchester UK
- 9. v To Dan, Edward and William with love.
- 10. Preface One of the major challenges currently facing the scientific community is to understand the function of the 20,000–25,000 protein-coding genes that were revealed when the human genome was fully sequenced. This book details the transgenic techniques that are currently used to modify the genome in order to extend our understanding of the in vivo function of these genes. Since the advent of transgenic technologies, the mouse has become by far the most popular model in which to study mammalian gene function. This is due to not only its genetic similarity to humans but also its physiological and, to a certain extent, its anatomical similarities. Whilst a large proportion of this book is dedicated to the use of the mouse in transgenesis, the mouse is certainly not the only model to provide essential information regarding gene function. A number of other valuable models are used in transgenic studies including Drosophila, C. elegans, Xenopus, zebrafish, and rat. For each of these species, a chapter in this book is dedicated to highlighting how each is particularly suited, for example, to the study of embryonic development, physiological function of genes and to study orthologs of human disease genes. These chapters give detailed practical descriptions of animal production, construct design, and gene transfer techniques; recently developed methods will be described along with highly established classical techniques. A number of chapters in this book are dedicated to the generation of genetically modified mice by the present classic techniques of injection of exogenous DNA into the pronuclei of fertilised eggs and by gene targeting using homologous recombination in embryonic stem cells. These chapters, as with all the others in the book, have been specifically written for this edition of Transgenesis and so contain up-to-date details of the practices in the field. Chapters are included describing optimal transgene and construct design, in-depth technical details for pronuclear microinjection of transgenes and associated surgical techniques, details for the optimal conditions in which to culture embryonic stem cells in order to maintain their pluripotent state, and methods for targeting these cells. A combination of chapters (Chaps. 13–15) describe how to generate chimaeras by microinjection of targeted ES cells into blastocysts or by morula aggregation, and the surgical techniques required to transfer the resulting embryos. For a number of years, the use of Cre/loxP and flp/frt recombination systems has gained in popularity; Chap. 16 describes their use and introduces other state-of-the-art site- specific recombination systems that can be used to manipulate the mouse genome. The generation and use of Cre-expressing transgenic lines are described in Chap. 17. One chapter of the book highlights the large-scale international efforts that are being made to systematically knockout every gene in the genome. The remaining chapters detail the breeding and husbandry skills required to successfully propagate a transgenic line and the increasingly essential methods for cryopreserving a mouse line and recovering lines from frozen stocks. This book is a comprehensive practical guide to the generation of transgenic animals and is packed full of handy hints and tips from the experts who use these techniques on a vii
- 11. viii Preface day-to-day basis. It is designed to become an invaluable source of information in any lab currently involved in transgenic techniques, as well as for researchers who are newcomers to the field. This book also provides essential background information for scientists who work with these models but have not been involved in their generation. On a personal note, it has been a great pleasure to edit this latest edition of Transgenesis. Firstly, I learnt many of my skills from reading earlier editions of the book and I hope that this edition will help and inspire many others. Secondly, I have been privileged to work with the exceptionally talented researchers in the transgenesis field who have contributed to this book. Manchester, UK Elizabeth J. Cartwright
- 12. ix Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I TRANSGENESIS IN VARIOUS MODEL SYSTEMS 1. Transgenesis in Drosophila melanogaster. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Leonie Ringrose 2. Transgenesis in Caenorhabditis elegans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Matthias Rieckher, Nikos Kourtis, Angela Pasparaki, and Nektarios Tavernarakis 3. Transgenesis in Zebrafish with the Tol2 Transposon System . . . . . . . . . . . . . . . . . 41 Maximiliano L. Suster, Hiroshi Kikuta, Akihiro Urasaki, Kazuhide Asakawa, and Koichi Kawakami 4. Generation of Transgenic Frogs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Jana Loeber, Fong Cheng Pan, and Tomas Pieler 5. Pronuclear DNA Injection for the Production of Transgenic Rats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Jean Cozzi, Ignacio Anegon, Valérie Braun, Anne-Catherine Gross, Christel Merrouche, and Yacine Cherifi PART II TRANSGENESIS IN THE MOUSE 6. Cell-Type-Specific Transgenesis in the Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 James Gulick and Jeffrey Robbins 7. Transgene Design and Delivery into the Mouse Genome: Keys to Success . . . . . . 105 Lydia Teboul 8. Overexpression Transgenesis in Mouse: Pronuclear Injection . . . . . . . . . . . . . . . . 111 Wendy J.K. Gardiner and Lydia Teboul 9. Gene-Targeting Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 J. Simon C. Arthur and Victoria A. McGuire 10. Gene Trap: Knockout on the Fast Lane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Melanie Ullrich and Kai Schuh 11. Culture of Murine Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Ivana Barbaric and T. Neil Dear 12. Targeting Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Roland H. Friedel 13. Generation of Chimeras by Microinjection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Anne Plück and Christian Klasen
- 13. x Contents 14. Generation of Chimeras by Morula Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . 219 Anne Plück and Christian Klasen 15. Surgical Techniques for the Generation of Mutant Mice . . . . . . . . . . . . . . . . . . . 231 Anne Plück and Christian Klasen 16. Site-Specific Recombinases for Manipulation of the Mouse Genome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Marie-Christine Birling, Françoise Gofflot, and Xavier Warot 17. Cre Transgenic Mouse Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Xin Wang 18. Large-Scale Mouse Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 Elizabeth J. Cartwright 19. Dedicated Mouse Production and Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Lucie Vizor and Sara Wells 20. Biological Methods for Archiving and Maintaining Mutant Laboratory Mice. Part I: Conserving Mutant Strains . . . . . . . . . . . . . . . . 301 Martin D. Fray 21. Biological Methods for Archiving and Maintaining Mutant Laboratory Mice. Part II: Recovery and Distribution of Conserved Mutant Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Martin D. Fray Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
- 14. Contributors IGNACIO ANEGON • INSERM – Institut National de la Santé et de la Recherche Médicale, Nantes, France J. SIMON C. ARTHUR • MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK KAZUHIDE ASAKAWA • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan IVANA BARBARIC • Department of Biomedical Science, University of Sheffield, Sheffield, UK MARIE-CHRISTINE BIRLING • Institut Clinique de la Souris – Mouse Clinical Institute (ICS-MCI), Illkirch, France VALÉRIE BRAUN • genOway SA, Lyon, France ELIZABETH J. CARTWRIGHT • Cardiovascular Medicine, University of Manchester, Manchester, UK YACINE CHERIFI • genOway SA, Lyon, France JEAN COZZI • genOway SA, Lyon, France T. NEIL DEAR • Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, UK MARTIN FRAY • Frozen Embryo & Sperm Archive (FESA), Medical Research Council, Mammalian Genetics Unit, Harwell, UK ROLAND H. FRIEDEL • Institute of Developmental Genetics, Helmholtz Center Munich, Neuherberg, Germany WENDY J.K. GARDINER • Mary Lyon Centre, Medical Research Council, Harwell, UK FRANÇOISE GOFFLOT • Institut Clinique de la Souris – Mouse Clinical Institute (ICS-MCI), Illkirch, France ANNE-CATHERINE GROSS • genOway SA, Lyon, France JAMES GULICK • Molecular Cardiovascular Biology, Cincinnati Children’s Hospital, University of Cincinnati, Cincinnati, OH, USA KOICHI KAWAKAMI • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan HIROSHI KIKUTA • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan CHRISTIAN KLASEN • Transgenic Service, European Molecular Biology Laboratory, Heidelberg, Germany NIKOS KOURTIS • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece JANA LOEBER • Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany xi
- 15. VICTORIA A. MCGUIRE • MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, UK CHRISTEL MERROUCHE • genOway SA, Lyon, France FONG CHENG PAN • Vanderbilt University Program in Developmental Biology and Department of Cell and Biology, Vanderbilt University Medical Center, Nashville, TN, USA ANGELA PASPARAKI • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece TOMAS PIELER • Department of Developmental Biochemistry, University of Goettingen, Goettingen, Germany ANNE PLÜCK • Centre for Mouse Genetics, Institute for Genetics, University of Cologne, Cologne, Germany MATTHIAS RIECKHER • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece LEONIE RINGROSE • IMBA – Institute of Molecular Biotechnology GmbH, Vienna, Austria JEFFREY ROBBINS • Molecular Cardiovascular Biology, Cincinnati Children’s Hospital, University of Cincinnati, Cincinnati, OH, USA KAI SCHUH • Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany MAXIMILIANO L. SUSTER • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan NEKTARIOS TAVERNARAKIS • Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece LYDIA TEBOUL • Mary Lyon Centre, Medical Research Council, Harwell, UK MELANIE ULLRICH • Institute of Physiology I, University of Wuerzburg, Wuerzburg, Germany AKIHIRO URASAKI • Division of Molecular and Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka, Japan LUCIE VIZOR • Medical Research Council, Harwell, UK XIN WANG • Faculty of Life Sciences, University of Manchester, Manchester, UK XAVIER WAROT • EPFL FSV – École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland SARA WELLS • Medical Research Council, Harwell, UK xii xii Contributors
- 16. Part I Transgenesis in Various Model Systems
- 17. Chapter 1 Transgenesis in Drosophila melanogaster Leonie Ringrose Summary Transgenesis in Drosophila melanogaster relies upon direct microinjection of embryos and subsequent crossing of surviving adults. The necessity of crossing single flies to screen for transgenic events limits the range of useful transgenesis techniques to those that have a very high frequency of integration, so that about 1 in 10 to 1 in 100 surviving adult flies carry a transgene. Until recently, only random P-element transgenesis fulfilled these criteria. However, recent advances have brought homologous recombination and site-directed integration up to and beyond this level of efficiency. For all transgenesis techniques in Drosophila melanogaster, microinjection of embryos is the central procedure. This chapter gives a detailed protocol for microinjection, and aims to enable the reader to use it for both site-directed inte- gration and for P-element transgenesis. Key words: Drosophila melanogaster, Embryo, Microinjection, Transgenic, Recombination, Inte- gration, Homologous recombination, phiC31/integrase, Site-directed integration, P-element Transgenesis in Drosophila melanogaster has undergone something of a revolution in the last few years. The classical technique of random P-element-mediated transgenesis has recently been sup- plemented by two novel technologies: homologous recombi- nation and ΦC31 integration (for reviews, see (1) and (2)). In P-element transgenesis (3), a modified transposon vector is used in combination with transient expression of the P transposase enzyme to generate several fly lines with different insertion sites in the genome. These insertions are subsequently mapped and characterised. P-element insertions have been invaluable for mutagenesis screens, but until recently, this was also the only 1. Introduction Elizabeth J. Cartwright (ed.), Transgenesis Techniques, Methods in Molecular Biology, vol. 561 DOI 10.1007/978-1-60327-019-9_1, © Humana Press, a part of Springer Science+Business Media, LLC 2009 3
- 18. 4 Ringrose method available for introducing a transgene of choice into the Drosophila genome. The random nature of P-element insertions has several drawbacks for transgene analysis. Mapping of inser- tion sites is time consuming, and transgene expression levels are subject to genomic position effects, making it difficult to draw comparisons between different constructs. A recently developed alternative to random insertion is homologous recombination (4, 5). This involves inserting a donor construct at random into the genome by P-element trans- genesis, and in subsequent generations, mobilising the donor construct to the correct locus by homologous recombination. This technique had long been lacking to Drosophilists, but has not replaced P-element transgenesis as the method of choice for routine transgene analysis, because both the cloning of donor constructs and the generation of homologous recombinants are more time consuming than for P-element transgenesis. Recently, ΦC31 integration has been developed (6). This technique allows rapid and efficient generation of site-specific integrants, and relies upon ‘docking site’ fly lines, which carry a single recognition site (attP) for the phage ΦC31 integrase enzyme, previously introduced into the genome by P-element transgenesis. A donor plasmid carrying a second recognition site (attB) and a source of integrase enzyme is used to generate flies in which the donor plasmid docks to the genomic site. Integration events are highly specific, as the attP site is 39 bp long and does not occur at random in the Drosophila genome. Many mapped and characterised docking site lines are now available (see Note 1), and ΦC31 integration is rapidly becoming widely used for many transgenic applications. All these transgenic techniques rely upon microinjection of embryos as a first step. In early Drosophila embryogenesis, the nuclei share a common cytoplasm for the first nine divisions. Directly after the tenth division, the first cells to become sep- arated are the pole cells, which will later form the adult germ line. Transgenic animals are made by microinjecting DNA and a source of enzyme (P-transposase or ΦC31 integrase, see Note 2) into the posterior of the embryo where the pole cells will form, at an early stage before they have become separated from the common cytoplasm. DNA can enter the nuclei and is integrated into the genome of some cells. Embryos are allowed to mature and the adults are outcrossed to screen for transgenic flies in the next generation. This chapter gives a detailed description of microinjection, from preparing DNA to screening for transformants. The main protocol deals with ΦC31 integration as we perform it in our laboratory. Alternatives for both ΦC31 and P element transgen- esis are given in the notes.
- 19. Transgenesis in Drosophila melanogaster 5 1. Donor plasmid containing attB site and transgene of interest (see Note 3). 2. Helper plasmid expressing ΦC31 integrase (see Note 2). Midi- or miniprep kit for preparation of plasmid DNA (Qiagen). 3. Absolute ethanol. 4. 3 M NaOAc, pH 5.2. 5. Sterile distilled water. 1. Capillaries: borosilicate glass capillaries, 1.2 mm × 0.94 mm 2. Needle puller: P-97 micropipette puller (Sutter instruments). 3. Needle grinder: Narishige microgrinder EG-400. 1. Fly line containing genomic attP site (see Note 1). 2. Fly bottles. 3. Fresh yeast paste: cubes of fresh baker’s yeast cubes are obtain- able from large supermarkets. They can be frozen and stored at −20°C for several months. Thaw at room temperature and mix with a little water to give a thick paste. 4. Dried yeast: Mix instant yeast granules with water to give a thick paste. Both fresh and dried yeast paste can be kept at 4°C for up to a week. Do not seal the container tightly, as the paste will expand. 5. Fly cages: PVC plastic tubing of either 50 mm or 90 mm diameter is cut into 100–150 mm sections and sealed at one end with nylon or metal mesh. The other end fits onto to a 50-mm or 90-mm agar plate, which is taped in place for egg collection. 6. Agar plates: Add 18 g agar to 600 mL tap water and bring to boiling point by microwaving. Dissolve 10 g sucrose in 300 mL tap water, heating a little if necessary. Add the sucrose solution to the agar, add 3.5 mL 100% acetic acid and mix well. Pour into petri dishes (90 mm or 50 mm) and allow to cool. Store for 1 day at room temperature to dry before using. Plates can be stored wrapped in plastic at 4°C for several weeks. About 16–20 plates per day of injection are required per cage (see Note 5). 1. Filtration apparatus consisting of glass funnel, filter support, stopper, sidearm flask, and clamp, suitable for 50-mm mem- brane filters. Attach the apparatus to water tap as shown in Fig. 1. 2. Materials 2.1. Preparation of DNA 2.2. Preparation of Injection Needles (see Note 4) 2.3. Preparation of Flies for Egg Laying 2.4. Dechorionation and Dessication of Embryos
- 20. 6 Ringrose 2. Bleach solution: mix 50 mL household bleach (2.8% hypochlorite) with 50 mL sterile distilled water. Make fresh every day. Wear a lab coat and gloves when handling bleach, as it bleaches clothes upon contact and is harmful to skin. 3. Membrane filters: mixed cellulose ester membrane filters, black with white grid marking. Circular, 50-mm diameter, 0.6-μm pore size (Schleicher and Schuell, type ME 26/31 ST). 4. Binocular dissection microscope. 5. Fine stiff paintbrush with nylon hairs: cut away hairs until only a few remain, for use in aligning embryos. 6. Dissection needle. 7. Forceps. 8. Microscope slides: use slides with frosted part for labelling, such as Superfrost plus (Fisher). 9. Coverslips: 24 × 24 mm. 10. Embryo glue: Make three balls of 2.5-m Scotch tape Magic 810 (3 M). Add these to 30 mL heptane in a 50-mL falcon tube. Shake vigorously at 28°C for 24 h. Cut a hole in the bottom of the falcon tube and drain solution into a small glass bottle. This glue keeps for several months at room tem- perature (see Note 6). 11. Drying chamber: 150-mm petri dish containing orange self- indicating silica gel granules: check that the silica gel gran- ules are orange; if they are not then they are saturated and no longer effective for drying embryos. Change to fresh granules. 12. Halocarbon oil: Voltalef 10S halocarbon oil, or halocarbon 700 oil (Sigma). Fig. 1. Filtration apparatus.
- 21. Random documents with unrelated content Scribd suggests to you:
- 22. awoke, then with a well-directed kick sent him flying forward, where that meek individual fell upon his knees and prayed, "that the devil might be cast out of that good man, the boatswain." Having vented his rage upon the soldier, Shever took out a bottle, filled a glass with rum, and drank the health of Captain Crushe, "and may all such duffers as some people perish," and from that day shunned the spirit of faith according to private Silas Bowler, and clave only to that more potent spirit yclept rum.
- 23. CHAPTER XVII. Upon finding himself comparatively free, Thompson's spirits rose, and he chatted with his guards in a most affable manner. After giving him to understand that if he made any attempt to escape they would strangle him, he was allowed to untie his rope-collar and carry it wound about his body, under his clothes. At night they stopped at the residence of a military mandarin, who billeted them upon the keeper of a tavern, their order running as follows:— "You Teen, keeper of the house of entertainment for travellers called 'The abode of ten thousand satisfied desires,' are directed to afford lodging and food to two imperial soldiers named Yung and Pang, and their prisoner Kwo-chau-ho-che, given on the ninth day of the tenth moon, &c., &c. Respect this. "(Signed) Han, "Second assistant governor." Pang, who was a sort of corporal, read the chop or order, then observed with the greatest complacency, "that the fleas of Teen's establishment were larger and more fierce than any others in that part of China," upon which Yung retorted that "they must be large and powerful, to be able to bore through such a tough skin as Pang's," and with many other merry observations the soldiers beguiled the journey until they arrived at "The abode of ten thousand satisfied desires," which turned out to be a dirty little inn, situated outside the walls, near the execution ground. Yung purchased a small portion of opium, and procuring a pipe from Teen, was soon in a state where all prisoners are free. Pang, who pretended to be very much disgusted, thereupon enjoined his prisoner to keep an eye upon his comrade, and retired to an up- stairs room, where he indulged in a debauch of warm rice-spirit.
- 24. Jerry mingled with the guests, and soon found the place was a notorious lodging-house for thieves and low characters. As the soldiers were both fast asleep. Teen had them conveyed to a dirty cell in an outbuilding; and knowing Jerry was their prisoner, directed him to be accommodated with a mat in the same apartment. About ten o'clock a woman brought them a bowl of rice, and a pot of tea, upon which the prisoner supped, and by eleven o'clock all the night-lights of the establishment were extinguished, except the one in the cell occupied by the soldiers and their prisoner. Finding they were both too far gone to resist, the sailor first secured their wrists and ankles, then laying them side by side, lashed them together, in the same manner as he would have done a hammock. After gagging them, he opened the door and walked into the inn. The dogs, aroused by his entry, began to growl and bark, upon which a watchman arose, and having rubbed his eyes proceeded to open a door, imagining he had heard some one knocking for admittance: seeing this the sailor quietly slipped through, and found himself in the street. After walking for some time he began to feel weary, but knowing that if he did not get clear of the place by daylight some one might identify him, or notice his unshaven head, he kept right on, every now and then finding himself dozing as he walked. At daybreak he found he was ascending a range of hills, upon the slopes of which he observed large tea-plantations. Groups of girls crossed his path upon their way to gather tea, and some of them passed jocular remarks, or invited him to join them and assist in their labour. About seven o'clock he met a travelling barber who, for a few sapecks, shaved, trimmed, and shampooed him, that operation taking place by the roadside, and only attracting the notice of two or three children who were on their way to school. When Jerry had secured his guards, he had searched their persons, and removed the purse he found upon Corporal Pang; justifying this act upon the grounds that when he was arrested in Whey-chú, these same soldiers had plundered him of all his money, therefore he was
- 25. merely regaining his own. Having paid the barber, he proceeded into the country, stopping every now and then to refresh himself. By night he had travelled a good distance; so imagining himself safe, he entered a tea-house, and having supped, turned in with about forty other travellers, and enjoyed their society in company with a host of agile tormentors. The room was a spacious one, and at the upper end a fat-lamp was kept alight all night. Jerry could not sleep, not being iron-clad like his companions, so he sat up and took a survey of the place. It was amusing to watch the features of the sleepers, who, unmindful of the ticklers, were snoring in a great variety of keys. At times, however, when their tormentors pulled rather too savagely, a solemn oath would issue from the sleepers' lips; and upon one occasion a savage-looking Tartar, roused by the bite of some patriarchal and artful Pulex, kicked the person who was sleeping by his side. The gentleman thus assaulted was reclining with his face towards his assailant, and as he received the kick in his waist, he was completely doubled up by the blow. After remaining quiet for a few moments, the fellow opened his eyes, and being a peaceful Chinaman, upon finding the person who kicked him was a Tartar, quietly turned over, as much as to say, "Now batter away if you will," but he declined to remonstrate with the person who kicked him. Not that he acted in this inoffensive manner from want of feelings or usually "when his brother smote him upon the left cheek, offered him his right." Had it been a Chinaman weaker than himself who thus assaulted him, he would have very soon retaliated, but the Tartar's savage face and burly form rendered him as quiet as a lamb. Thompson was highly amused with the performance; so, picking up a straw, he proceeded to tickle the Tartar. For a long time the man bore it, probably the irritation not amounting to much; however, at last, upon the sailor thrusting the straw up his nose, he lifted his foot and again kicked the Chinaman, who thereupon assaulted the celestial next to him, and he in return favoured his companion. A tremendous row ensued, upon which the landlord and his assistants rushed into the room, and laid about them with bamboos, until order was restored.
- 26. Long before daybreak they all cleared out, and the sailor, having partaken of a light breakfast of rice and tea, made for the hills. After going a short distance, he fell in with a party of tea-gatherers, who invited him to join them. As he had no definite plan for the future, he accepted their offer, and, receiving a basket, was soon toiling up the hill-side. The business was one which required the labourers to be at work by sunrise, as the kind of tea they were gathering is not picked when the sun gets too far up. A light fog hung about the hills, and the faces of most of the women were enveloped in wrappers, but as the day broke they took off these cloths, and revealed some very pretty countenances. Upon their arrival at the plantation to which the party were bound, the leader appointed the pickers and carriers: the former were expert young girls, who had been trained to the business from childhood, while the latter consisted of the "dull-heads," or men; and as the sailor was supposed to be a poor Cantonese, who could know nothing about picking tea, he was directed to hold the basket for a sprightly girl named A-tae. Now, it is usual for the girl who picks the finer kinds of tea to be dressed in much better clothes than her basket-holder, and as A-tae was a beauty, and tolerably well off, she was smartly attired; true, her garments were not very costly, but they were new and jauntily worn. Her dress consisted of two pieces, the usual loose blue trousers and wide-sleeved jacket, her hair being braided in queues which descended to her waist, while her head was protected from the sun by an immensely wide bamboo hat. When the overseer directed the sailor to bear her basket she had not cast eyes upon the latter, having been listening to the silly story of a companion, so, thinking it was the usual "dull-head," she waved him to follow her, and turned into one of the rows; then dexterously grasping a handful of leaves, she cried, "Come here!" and upon his placing the sieve-like basket under her hands, showered the leaves into it with marvellous rapidity. Having exhausted one bush, she was moving towards another, when, catching sight of her attendant, she
- 27. uttered a little scream, and coquettishly turned away her head. Seeing her agitation, the enamoured basket-holder inquired if she were unwell. "No! I'm—Come here, you fright!" The girl worked like lightning, ordering her holder about in a most imperious manner. At last curiosity overcame her, and she demanded the name of her slave. "I have no name." "No! How shall I call you, then?" "Call me Sa" (ugly of the sort). "Oh no! oh no; that would be cruel." "Call me Cha-tee" (a mean fellow). "No, no, for you are not mean." "What will you name me, then?" said Jerry, looking as though he could devour her. "What you call me shall be my name." A-tae trembled, as she cast a timorous glance towards her basket- bearer, and replied, "I call you Sho" (beautiful eyes), saying which she laughed, and added, "but surely you will not take that name?" "I'll call myself any thing you choose to name me." "Then I give you this,—Yung-Yung" (good-humoured face). And what may I call you?" "Me! Don't you know?" said the pretty girl, looking at Yung-Yung in a manner which made his heart bump again. "What! not know my name?" "I do not. I am a wanderer and a stranger here."
- 28. "Poor fellow. Have you no friends?" "None here. Will you be my friend?" "You don't know my name, yet ask me to be your friend. Speak lower, and look down while you talk, or the overseer will send some one else with me to-morrow." "What is your name?" "A-tae." After casting his eyes about in order to ascertain if any of the pickers were watching, he bent over the girl, who was very deeply engaged in removing some fine shoots from the lower part of a plant, and when she rose, as her cheek came quite close to his, he kissed it gently, and said, "A-tae, I love you." The girl gave a nervous little laugh, then asked him what he meant. "I want to marry you." "Where do you come from, Yung-Yung-Sho, that you speak thus? Would I could be given to one like you; but I shall be, like other girls, sent off to slave for some man of my own class, or sold to a mandarin." (It will be perceived that A-tae was, although a Chinese, an advocate for woman's rights). "Oh, Yung-Yung-Sho do you think Buddha knows how badly they treat us poor girls?" "Can't you run away with me?" observed the now thoroughly "gone" sailor; "slip off in the night, and go away to a country where the women are thought as much of as the men." "That's where Buddha is, Yung-Yung-Sho. There we shall be men. I know all about that, and have my Tieh papers at home. I'm not as stupid as most girls. You are a benevolent man thus to listen to the nonsense of little me. But why do those Yuen-chae (police runners)
- 29. point this way? Are you wanted? If so, flee. That way, that way; up among the rocks, and hide in the caves." Jerry had little time to say farewell, as he noticed the two soldiers, accompanied by police runners, making towards him; so, after bestowing a fervent kiss upon the lips of the astonished A-tae, he sprang over the tea plants and sped away like the wind. The poor girl sunk upon the ground, cried, and wrung her hands like one demented. Her companions gathered round, and finding she was in trouble, prevailed upon her to go home. Meanwhile the soldiers and their party chased the agile sailor, running until they got out of breath; and when they last spied him he was darting into a wood, which was set apart for the use of Buddhist priests, and where they felt sure of bagging him during the course of the day. A-tae walked home like one in a dream, and was questioned by her mother, who anxiously inquired if she had "seen a spirit," she looked so scared and pale. She had seen one, the recollection of whom would never again be absent from her mind. She was in love, had been spoken to by a being, one of the opposite sex, who neither commanded nor treated her like an inferior animal. Was it a dream? Was he not one of those genii who, assuming the appearance of gods, use their fatal beauty to destroy all whom they fall in with? What could he be? Poor little girl! She was sorely tried; so taking a few sticks of incense, she burnt them before the picture of the Kitchen god, in order if possible to get him on her side. But she didn't tell her mother about Yung-Yung-Sho. Towards the evening she became very ill; and by night her anxious parents sent for a doctor, who, after writing a prescription, submitted it to them. "How much will it cost?" demanded the father. "Two hundred cash," gravely replied the man of physic.
- 30. "Can't you do it a little cheaper? we are poor people." "I don't think I can. Let me see. I can leave out the dried rats' tails— they are costly—and the alligator's blood may be omitted. Well, say one hundred cash." The mother was a clever women, and didn't believe in the doctor's nostrum's, so she demanded how much the gentlemen wanted for the prescription. "Fifty cash." "Pay him and let him go, my lord," she observed to her husband, who thereupon handed over the cash, and the doctor departed. When he was out of sight the old woman nodded shrewdly towards her husband, as much as to infer, "trust me for being smart," then having prostrated herself before the picture of the Kitchen god, gravely burnt the prescription, and pouring some warm tea upon the ashes, carried the drink to her daughter, and compelled her to swallow it, saying soothingly, "You'll be all right to-morrow." "Oh, my heart, my heart," moaned the poor little girl. "Oh, it is not your heart, A-tae, it's your brain that has become oiled by the sun. You'll be all right now, as it will congeal again;" and having delivered herself of this very Chinese opinion, the old lady withdrew, leaving the poor child to combat a disease as old as the hills, and for which there has never been but one cure since the world began. Nothing but the possession of the loved one will satisfy the poor souls, who, like A-tae, suffer from this awful affliction. No doctor can cure them,—possibly the priest may,—but not the man of medicine. When the girl's mother saw her husband the latter did not ask how fared his darling A-tae. She was but a girl, and her death would not cause him to shed a tear, but the mother made up her mind to one thing, as she informed her help. "If that girl gets a little better, I'll take her to Nan-woo," a very sanctified Buddhist bonze, who lived in
- 31. a hole in a rock situated in the Buddhist grove, distant about eight li from her house. But A-tae became worse, so they bled her. This took away what little strength she had left, and the gossips said she would soon salute heaven. Upon the afternoon of the fifth day some of the women round her bed were speaking about the hunt after the stranger who had been working with A-tae upon the day she was taken sick, and after observing that "he must have bewitched the child," they mentioned something which had a wonderful effect upon the girl, and which caused her to rally from that moment. Jerry, having distanced his pursuers, determined to search for the caves of which A-tae had spoken. There was little difficulty about the matter, as the rocks were full of them; so having found one which he thought would suit, he quietly stretched himself upon the floor and went to sleep. As there was nothing to encourage the presence of the pulex family, he slumbered without annoyance. After dreaming of A-tae, and imagining they were about to united at the altar, with Mr. Shever acting as best man, and Miss Pferdscreptern as bridesmaid, Mary Ann being present in charge of a small family of Chinese children, one of whom strongly resembled Captain Puffeigh, the bewildered sailor woke, and upon rubbing his eyes, discovered that he was being watched by one of the police runners, who, when he saw him open his eyes, gave a loud alarm. Jerry got up, stretched his limbs, and then, walking to the entrance, took a critical survey of his position. The cave was dug out of the limestone rock and was approached by two paths, while in front was a steep decline down which it was impossible to escape. Gazing to the left he saw Corporal Pang, supported by a police runner armed with a short sword, while approaching upon his right was private Yung, similarly assisted. Thompson whistled. Pang suddenly stopped, and called upon him to surrender. Yung bawled to him to give up at once, or he'd kill him when he got hold of him.
- 32. The undaunted sailor only whistled all the louder. Seeing he was quietly awaiting their arrival, as if determined to give himself up, the soldiers clambered up the hill until Yung who was nearest him, stopped to breathe, upon which Thompson rushed at him, bowled him over like a ninepin, floored his attendant with a blow in the chest and then darted down the pathway and disappeared from sight; and Pang arrived at the top of the hill to find his companion in arms hors de combat. Yung being picked up by his comrade, and having acquainted him with the particulars of the assault, they again set off in search of the troublesome western devil. It was a smart chase, as the runners knew every inch of the ground; and after having sighted him several times, but to lose him again the next moment, one of them saw him disappear up a sort of ravine, from which they were certain he could not escape. "It is the retreat of Nan-woo, a very holy bonze, and he is as safe in that hole as a rat is in a bottle," observed one of the police. "He is a wizard, and will fly out if all other means fail him. Oh, I know we shan't catch him," grumbled Yung. "How can we fail, your excellency?" replied one of the attendants. "That path leads to a high rock, in which is a small hole, where Nan- woo entered fifty years ago. On each side of the path is a precipitous rock, which no man can climb; therefore, your foreign devil, upon finding the path leads to nowhere will retrace his steps. Let us, therefore, crouch down upon either side of the rocks at the entrance, place a cord across the pathway, await his return, and when he arrives we will lift the line, and trip him up." "Capital, capital!" cried the soldiers. Thereupon the party divided, and crouching down behind the gigantic boulders which lay beside the entrance to the gulch, string in hand awaited the return of the sailor. They calculated he would possibly have a little chat with the bonze, then, finding there was no other outlet, would fall into their hands, and be captured without difficulty. Every now and then some noise, probably caused by rabbits, would make them start and clutch
- 33. their line, but after waiting a considerable time, hunger reminded them that they had started upon the expedition without taking breakfast, and they determined to proceed up the ravine, and boldly bring the "eccentric one" to bay. Having explored nearly the entire length of the place, they turned a bend in the pathway, and found themselves before the retreat of Nan-woo; but where was the sailor. "I expect he is in there along with the bonze," whispered Yung. "Bosh! How could he get in there? Why, it is five feet from the ground, and the hole is too small." "Ask the hermit if he has seen a man?" put in one of the runners. Upon this Pang, who did not believe in Buddhism, and consequently had little respect for its bonzes, advanced to the opening, and rapping his sword handle against the screen, demanded if the old gentleman inside had seen a fellow trying to climb up the rocks which surrounded his cell. Fumbling at the slab of limestone which formed the screen before the entrance or pigeon-hole of his cell, repeating as he did so the words "o-mi-tu-fuh, o-mi-tu-fuh," the old bonze at last succeeded in pushing the panel into a hole, cut out for its reception in the side of the rock, and then asked, the soldier what he wanted, upon which the latter repeated his question. The old bonze looked at his interrogator for some moments; at length appearing to understand him, replied, "My son, since first I entered this abode, these eyes have never beheld a man attempt to scale those rocks—o-mi-tu-fuh, o-mi-tu fuh." "Come along, Pang; he's cracked. Let us seek the fellow in some other place; or, better still, we will return, or join the first party of rebels we come across, as it will never do for us to go back to our native town, and say we have lost him."
- 34. After a strict search they gave the matter up, and dismissing the police runners, proceeded to the nearest rebel town, where they were received with open arms by Ma-chow-wang, who commanded the insurgents in that district. When the sailor entered the ravine, he imagined it had another outlet, but upon discovering the small oven-like opening in the rock at the end (the same being open at the time), he, taking it for the entrance to a burial vault, after running to give himself impetus, sprang up, clutched the ledge with his hands, then forcing in his head and shoulders, wriggled through, and dropped upon the floor. Nan-woo was slumbering, but in his sleep repeating the words "o- mi-tu-fuh;" upon which Jerry shook him, then prostrated himself, and, to the best of his ability, repeated the same words to the astonished bonze, who looked at him with horror, and quaveringly demanded who he was. "O-mi-tu-fuh; o-mi-tu-fuh!" ejaculated the prostrate sailor. However, at length he got up, and, in his best Chinese, prayed the bonze would save his life, and hide him from his enemies. Nan-woo was a merciful old fellow; and as he had long desired an assistant, or disciple, agreed to shelter the fugitive. Having instructed him to hold his tongue, the old bonze took his position behind the screen, and awaited the arrival of the soldiers; how he got rid of them has been described. When night came the old fellow lit a lamp, and Thompson had an opportunity of seeing what his quarters were like. The cell was an irregular apartment, cut out of the solid limestone rock. There was no furniture, but an old mat, while a water jar, and an earthen chatty, containing a few handsful of dry rice, were the only kitchen articles the bonze possessed. Jerry surveyed the latter for a few moments, then asked if that was what he lived on? upon which the old man nodded, and taking a
- 35. handful of rice, threw a few grains into his mouth, then drank a sup of water. "Well," exclaimed the sailor in his native language, "here's a go. I've been and signed articles to a toad in a hole, and got to live in a box office, on dry rice and water." Their frugal meal having been partaken of, the old fellow chin- chinned his disciple, and with the assurance that no man would dare come up the gully at night (as he had declared it was haunted), the old gentleman dropped down upon his knees, and o-mi-tu-fuh'd at such a rate, that Jerry set it to music, and joined in a sort of chorus. "I wonder what the deuce it means? I used to hear poor Jow a saying of it. O-mi-tu-fuh (stretching himself, and yawning); don't I wish I had a tooth full of grog." When the sailor awoke the next morning he found the old bonze still at it,—"o-mi-tu-fuh, o-mi-tu-fuh!" and he kept it up all day, repeating the words in a mechanical sort of manner, which at times greatly irritated his companion. About ten o'clock a woman came, and asked what she should do to obtain luck. "Bring a dish of boiled rice and some tea, and place them in the road before my cell, as an offering to the evil spirits. Do this daily for a week." When she had departed another arrived, and the sailor amused himself, and improved his knowledge of the language by listening to their wants. At last one came whose story caused the man to be all ears. It was A-tae's mother, who thus detailed her daughter's symptoms. "She has devils in her brain, who speak for her, and I fear she will die."
- 36. Nan-woo, who had great faith in a youthful constitution, gave the afflicted mother two slips of bamboo, upon one of which was written, "Decline present benefit, and receive greater reward in future," while the other ran as follows; "Ten thousand devils are not as tormenting as a bad heart." A-tae's mamma read these, and accepted them as the words of an oracle, of course torturing their meaning to suit her daughter's case. "When A-tae gets well, what shall she do?" "Bring me every morning, for one month, a basket of fruit and some young tea, then I will assure her perfect health." Jerry gave a sigh of relief. "I'll see her again somehow," he thought. It was a few days after this that the gossips were chatting around A- tae's mat, and the following is what they said: "Oh, Mrs. So-and-so, have you heard the news? You remember how two soldiers hunted the man who frightened this poor child so? Well, they chased him to Nan-woo's hermitage, and the bonze told them as soon as the thing saw him it burst into a flame and vanished." "Did you ever?" cried one gossip. "Bless us!" said another. And little A-tae winked behind their backs. "Oh, splendid Yung-Yung-Sho, I shall see you again, my lord, my emperor, my deity. I shall live if I can only look upon you now and then. We will be like the Neih, who enjoy sublime love by merely glancing at each other. O dazzling Sho! You shall be my god, and I will burn incense to you day and night. My whole frame thrills with exquisite delight when I hear your voice. My eyes light up like lamps at night when I view you, Sho. Oh, my absorbing god, never look coldly upon A-tae. You will always speak gently to me, will you not? Always be so kind and tender to your little A-tae, who loves you from your queue to your shoes." Thus apostrophized the happy girl, and it
- 37. was no wonder old Nan-woo's charms worked, for Cupid was directing them; and as musk overpowers every other odour, so, beside love, all pleasures in this life are utterly dwarfed and lost. 'Twas love nearly caused the death of A-tae, and the same potent spell restored her to life and hope. "Now, whether you like it or not, you shall visit Nan-woo next week," observed the girl's mother. "I'll try," dutifully replied A-tae. "I'll go, mother, even if it kills me. I'd rather die than displease my parents." Cunning little A-tae!
- 38. CHAPTER XVIII. "Having received information that a notorious pirate, named Yaou- chung (short-tailed ruffian) is operating upon the coast between Chusan and Amoy, you are hereby ordered to proceed from Chinhae (where it is expected you will receive this dispatch), and carefully examine the coasts, particularly about Hae-tan Island. In the event of your capturing the pirate, you are directed to deliver him to the Taontai of Amoy, who will dispose of him as he sees fit, the pirate having a short time since seized a passenger junk, on board of which were fourteen mandarins belonging to that place, whom he enclosed in an iron cage and burnt alive. As we wish to show our power in these seas, it is desirable that you totally exterminate the band, and level their settlement to the ground." "A very nice little job, is it not, Russell?" observed Woodward, who had just received the above dispatch from the admiral at Hong- Kong. "As you most logically observe, sir, the occupation does most fully merit the title you so aptly apply to it, of a nice little job, and it will be as well to attempt the matter without procrastination." "There, there, my dear Russell, why not say we've got to do it, and will do it well?" "That, sir, would, no doubt, be a concise manner of expressing it, but I prefer to adorn my language with more classical and florid expressions." Upon hearing this reply, the good-tempered captain nodded to his eccentric lieutenant, and directed the ship to be got ready for sea. In a short time the anchors were up, and the Stinger steaming towards Hae-tan, every one being upon the qui vive, and anxious to fall in
- 39. with the notorious pirate. As Woodward anticipated some warm work when he met the freebooter, he ordered all useless top-hamper to be stowed below, the top-gallant yards and masts struck, and rigging snaked, intending to use steam alone in his trip down the coast. After a careful examination of the coast, and hearing some horrible tales of the cruelties perpetrated by Yaou-chung, Woodward arrived off Hae-tan at dusk one evening; and having slowly steamed across to the main land, anchored until daylight the next morning. About five bells in the middle watch, some junks passed, when he quietly turned out his men, not a sound being allowed or light shown, and the crew learnt that the piratical fleet was sailing in, and that by daybreak an action was inevitable. It was impossible to distinguish the junks with the naked eye, but with his night glass, Mr. Beauman made out nine large vessels, on board of which the Chinese, unaware of the presence of an enemy, were firing crackers and beating gongs in a most unguarded manner. When they were out of hearing, Captain Woodward got up anchor, and hugging the land, crept after them, and at daybreak saw the last of the fleet put up its helm and run into port. In a few moments the Stinger was tearing away at full speed for the place, the men watching their captain, who, assisted by the master, manœuvred the ship splendidly; and although the odds were eight to one, no one doubted his ability to do all he might undertake. Every one seemed impressed with a consciousness of responsibility, and appeared fully determined to do his duty; and when the ship swept round the point, and they found themselves in the entrance of a large bay, which was studded all over with junks, although they felt inclined to cheer, they held their peace, knowing, by the eyes of their commander, that they must repress their enthusiasm. Woodward stood upon the bridge, glass in hand, and gave his orders as calmly as he would have done had he been entering Hong-Kong harbour. At last he suddenly rang upon the engine-room bell the signal to "stop her," but before they could do this the ship struck
- 40. upon a mud bank, and at that moment the pirates sighted her, and altering their course, turned back and opened fire. It was a trying time: the vessel swinging across the passage, and forming as it were a target for their guns. After a while the junks suddenly ceased firing, and bout ship, when, having sailed some distance up the bay, they formed in two lines, and again bore down towards the Stinger, the execution of this manœuvre occupying about three-quarters of an hour. Having in vain tried to steam off and after running his crew backward and forward upon the upper deck, Woodward ordered the foremost guns to be transported aft, and then repeating his tactics, found the ship once more floated, whereupon the guns were returned to their proper positions, and they awaited the arrival of the pirates, who were about a mile distant. Upon her starboard bow were five large junks, the foremost of which was doubtless the flagship, it being beautifully painted and gilded, while on the port bow were four smaller craft letting off crackers, and making a great din with their gongs. When their guns arrived within range they commenced firing their bow chasers, Woodward surveying them through his glass as coolly as though they were performing their evolutions for his amusement. The shot flew over the Stinger, and now and then one would strike her hull, but there stood the captain quiet and undaunted, while his men, taking example from him, were as still as statues. Suddenly a heavy shot struck the funnel, near which he was standing, and cut a piece clean out of it, when he quietly lifted the handle of the engine-room bell, and rang out, "Go ahead, full speed," then waved his orders to the first lieutenant and master, stationed along the deck, who transmitted them to the men at the wheel. In a short time they reached the junks, but still no signal was given to fire, although the pirates were blazing away furiously, and some stray shots struck the hull and rigging. The men, who were all crouched down behind their guns, wondered when they were to commence, and now and then would peer over the pieces and watch
- 41. the unmoved commander. At last, just as they got abreast of the foremost junks, between which he had steered, the words "Commence firing" rang out from Woodward's lips, and at the same instant he signalled "Stop her" to the engineers. The men sprang up with a cheer of defiance, and poured a discharge of grape and canister into the junks on either side, (flash) bang (flash—flash—flash) bang—bang—bang—(flash) bang; and the excited sailors loaded and fired with tremendous energy. In a very short time a thick pall of smoke completely enveloped the ship, and with great difficulty the captain managed to keep her in position between the line of junks—she in the mean time drifting slowly ahead. After the first few discharges the men lost their hearing through the stunning reports, and would vainly bawl at each other, while their bodies were grimed with the smoke of the powder, every one of them being stripped to the waist. The powder-monkeys were as active as their namesakes, feeling their way in the thick smoke, so as to avoid being knocked down by the rammers or sponges, and cautiously treading clear of the tackle laid along the decks. It was wonderful how clever the youngsters were, and with what accuracy they would return to their own guns, although it was impossible to see them. The flashes, which at first dazzled their eyes, now merely made them blink for a moment, while their dulled ears only heard a faint boom, and after a time did not notice even that. Woodward sprang up aloft, and saw the ship was heading right, and that the first two junks which they had passed were on fire. Upon his return to the deck he met the master, who bawled something in his ear; but as he could not understand what he said,[1] he motioned him to go aloft, and keep a look-out. Although the Stinger steamed quite slowly between the lines of junks, she had not lost a man; and the pirates being unable to depress their guns sufficiently to hit the ship very often, had actually been firing into each other. When Woodward found that the shots were striking the ship in an oblique direction he rang the signal, "Go
- 42. ahead, full speed," and in a short time was clear of the junks, which, however, kept firing away at each other for some thirty minutes. After they discovered their mistake he came to anchor, and putting on a spring, raked them fore and aft with grape and canister. In a short time the two lines of junks closed upon each other; and as they were nearly all on fire, the pirates abandoned them, and took to the water. Much to Woodward's chagrin, he observed that the big junk, which he supposed was commanded by Yaou-chung in person, had managed to put out her fire, and was escaping through the passage to the sea; however, as it was impossible to pass the burning vessels, he steamed up the bay, and landed at a town about five miles from the entrance. The Taontai came down to receive him, and Woodward found that the pirates had that morning entered the place to collect tribute when they were overtaken and destroyed by the Stinger; and so grateful were the townspeople, or rather their governor, that he offered the ransom money to the captain, who of course declined the gift. Woodward did not want to risk his ship too near the burning junks, and he showed his prudence, for about 9 A.M. two of them blew up, and shortly afterwards the others followed; and as the explosions seemed to blow out every vestige of flame, they floated about the bay mere shapeless hulks, and became a prey to the swarms of thieves, who went out of the city in boats to pick up wood or any loot which they might be lucky enough to come across. Seeing the mouth of the bay clear, the captain bade the civil Taontai adieu, and steamed out to sea in search of Yaou-chung's junk. Upon clearing the headland at the mouth of the harbour they beheld the pirate with all sail set standing out to sea, but as soon as he saw them he trimmed his sails, and ran behind Haetan. Now, Woodward knew there was no shelter for the pirate upon the weather side of the island, so he altered his course, and steamed along to leeward, expecting to catch the junk as it rounded the opposite point; but Yaou-chung was too smart for him, as he had anchored, it being a
- 43. calm day, just round the point behind which Woodward saw him disappear. Having waited for two hours, the captain proceeded round the further point, and, to his annoyance, saw the pirate standing out to sea, with his sails so closely hauled, that he seemed to be going in the wind's eye. Now, every nautical writer has described a stern chase, and doubtless the old adage "A stern chase is a long chase" has been sufficiently hackneyed, but it was a very long one upon this occasion, as it must be remembered the Stinger was only an auxiliary screw, and it was quite dusk before they overhauled the plucky Chinaman. Woodward was at his post, and had given instructions to the master to lay the ship alongside the junk; and taking command forward of the starboard watch of boarders himself, instructed Lieutenant Russell to head those of the port watch, who were ordered to board the pirate abaft, directing the men to crouch behind the nettings until they struck the junk. Forward, the captain of the forecastle was securing the end of a chain, to which was fastened a grappling-iron, and abaft, the captain of the afterguard was similarly employed. The Stinger showed no light, and made no sound, save that caused by the regular beat of her screw. Suddenly the junk put about, and tried to rake the ship, but Woodward was too good a sailor to allow his enemy to catch him asleep, and the pirate threw his shot away upon the water. After various manœuvres, too tedious to describe here, the gallant captain at last got his ship in exactly the position he wanted her, and putting on full steam, ran her crash into the bows of the junk. Up sprang the captain of the forecastle, and the grappling-iron was firmly secured in the side hamper of the pirate, upon which Woodward shouting to his men, "Come on, my lads!" leapt sword in hand on board the junk, landing his party upon the forecastle, from which they drove the pirates with great slaughter. The Stinger was then laid alongside, and with a loud hurrah, Lieutenant Russell, led his men over the hammock-netting abaft, obtaining in a few
- 44. moments possession of the poop. The pirates, driven to the body of the junk, fought like demons, and twice repulsed the Stingers, once nearly recovering possession of the poop, which was, however, gallantly held by the first lieutenant. When Yaou-chung found he was cornered, he conceived the bold idea of trying to board the Stinger; so, giving instructions to his men, he, in spite of the shower of pistol-balls and musketry which was poured upon him from the poop and forecastle, succeeded in boarding the ship, before the master, who was in command, became aware of his manœuvre. Beauman was attending to the after grapnel, when he saw the pirates pour over the nettings just by the main hatchway. Without a moment's hesitation he darted below, ran forward upon the lower deck, sprang up the fore hatchway, and scrambling on board the junk, told the captain of the pirate's move. "All aboard!" shouted Woodward. Then directing two of the men to cast off the grapnel, he abandoned the junk, and drove the pirates aft upon the quarter-deck of the Stinger; the men who had cast off the grapnel on board the junk, running aft and telling the first lieutenant the news. Russell thereupon placed his men so as to cut the pirates down as they were driven off the ship's decks abaft. As all this was done upon a starlight night, the Stingers could just make out friends from foes, although at times the pirates and crew got a little mixed, and even assaulted their own shipmates. Yaou-chung led his men like a tiger, and certainly fought well; but just as he reached the wheel a light shot up on board the junk, and Woodward saw him motioning his men to press forward and attack the sailors again. With a loud cheer the Stingers threw themselves upon the foe, and their captain, wielding a cutlass which he had taken from one of his men, cut Yaou-chung down with a swinging blow. When the pirates saw their leader fall, they surrendered, and within a quarter of an hour seventy-three of them were secured and put in irons, together with Yaou-chung, the cut given by the commander having more stunned than otherwise injured him, his skull being thick enough to stand a chop from a cutlass.
- 45. When all was quiet, they carefully examined the prize, which was found to be filled with valuable plunder; then they threw the dead overboard, and taking her in tow, proceeded towards Amoy, where they arrived within eight-and-forty hours after the capture of the junk. The notorious Yaou-chung and his associates were duly handed over to the Taontai, after which the Stinger refitted and stopped up the shot-holes in her sides. She had been hulled eighteen times, but upon mustering her crew after the action, only thirteen casualties were reported, not one of which proved fatal. One man lost a limb, and another three of his fingers, but otherwise the wounds were slight. Of course it was by the merest good fortune they escaped as they did, for had the ship, when between the junks, been but for a moment in such a position that their shot could have taken effect, no doubt her decks would have been swept. It was a bold action, and the merchants of Amoy, to show their appreciation of Woodward's gallantry, offered him a service of plate, which he courteously, yet firmly, declined, alleging that his officers and men had quite as much to do with destroying the pirates as himself. He, however, did not object to their presenting his crew with a gratuity, which amounted to over three thousand dollars; and as he knew how slow the prize courts were, he told his men to clear out all they wanted from the junk, after which he despatched her to Hong-Kong, where she was condemned and sold. Not a bale of silk or ball of opium would he keep for himself, being too proud to share in the plunder; and beyond a few flags, taken from the various pirate junks he had destroyed, he returned home no richer than he came, his principles being totally unlike those of his predecessor, who upon one occasion, after taking a junk, coolly appropriated a number of balls of opium, which for security he stowed in the lockers of his state room, the said opium being described by him as "his perquisites." After remaining in port a few days, a grand banquet was given by the Taontai, to which the captain, officers, and crew were invited;
- 46. Welcome to our website – the ideal destination for book lovers and knowledge seekers. With a mission to inspire endlessly, we offer a vast collection of books, ranging from classic literary works to specialized publications, self-development books, and children's literature. Each book is a new journey of discovery, expanding knowledge and enriching the soul of the reade Our website is not just a platform for buying books, but a bridge connecting readers to the timeless values of culture and wisdom. With an elegant, user-friendly interface and an intelligent search system, we are committed to providing a quick and convenient shopping experience. Additionally, our special promotions and home delivery services ensure that you save time and fully enjoy the joy of reading. Let us accompany you on the journey of exploring knowledge and personal growth! ebookultra.com