Table Of ContentGGnnoottoobbiioottiicc
MMoouussee
TTeecchhnnoollooggyy
A N I L L U S T R A T E D G U I D E
GGnnoottoobbiioottiicc
MMoouussee
TTeecchhnnoollooggyy
A N I L L U S T R A T E D G U I D E
Chriss J. Vowles • Natalie E. Anderson
Kathryn A. Eaton
Boca Raton London New York
CRC Press is an imprint of the
Taylor & Francis Group, an informa business
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
© 2016 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business
No claim to original U.S. Government works
Version Date: 20150917
International Standard Book Number-13: 978-1-4987-3633-6 (eBook - PDF)
This book contains information obtained from authentic and highly regarded sources. Reasonable
efforts have been made to publish reliable data and information, but the author and publisher cannot
assume responsibility for the validity of all materials or the consequences of their use. The authors and
publishers have attempted to trace the copyright holders of all material reproduced in this publication
and apologize to copyright holders if permission to publish in this form has not been obtained. If any
copyright material has not been acknowledged please write and let us know so we may rectify in any
future reprint.
Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced,
transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or
hereafter invented, including photocopying, microfilming, and recording, or in any information stor-
age or retrieval system, without written permission from the publishers.
For permission to photocopy or use material electronically from this work, please access www.copy-
right.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222
Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that pro-
vides licenses and registration for a variety of users. For organizations that have been granted a photo-
copy license by the CCC, a separate system of payment has been arranged.
Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are
used only for identification and explanation without intent to infringe.
Visit the Taylor & Francis Web site at
http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
This work is dedicated to Gayle Francis:
helper, friend, and colleague.
Without you, this would not have been possible.
Contents
Preface and Acknowledgments .....................................................xiii
About the Authors ........................................................................xv
1 A Brief History of Germ-Free Life ............................................1
References ..............................................................................4
2 Overview of Gnotobiotic Technology ........................................5
What Are Germ-Free Animals? ...............................................5
How Do We Get Germ-Free Mice? ...........................................6
Germ-Free Housing and Barrier Types ...................................7
References ............................................................................10
3 Equipment and Terminology .................................................11
4 Personal Protective Equipment (PPE) ....................................19
Introduction .........................................................................19
Types of PPE .........................................................................20
Applying Sterile PPE .............................................................21
Supplies ..........................................................................21
Procedure .......................................................................21
5 Sterilants and Sterilization ...................................................27
Introduction .........................................................................27
Sterilization Methods ............................................................27
vii
viii Contents
Cold Sterilization ..................................................................28
Supplies ..........................................................................28
Procedure .......................................................................30
Wrapping Materials for Autoclaving ......................................32
Supplies ..........................................................................32
Procedure .......................................................................32
Gas Sterilization (Ethylene Oxide) ........................................34
Supplies ..........................................................................34
Procedure .......................................................................34
6 Isolator Setup .......................................................................39
Introduction .........................................................................39
Part 1: Small (5.5 × 3 feet) Isolator Assembly ........................41
Preparation of Isolator Components ................................41
Supplies ....................................................................42
Isolator Components ..................................................43
Preparing Hand-Wrapped Filters .....................................43
Supplies ....................................................................43
Installing the Isolator Components .................................50
Inflating and Sterilizing the Isolator ................................59
Decontaminate the Inside of the Isolator .........................64
Overnight Leak Test ........................................................66
Start the Isolator.............................................................66
Part 2: Large Isolator Assembly ............................................69
Introduction ....................................................................69
Supplies ....................................................................69
Preparation .....................................................................71
Sterilize the Filters ....................................................71
Sterilize the Air Supply Piping System .......................76
Prepare the Supplies .................................................76
Isolator Assembly ............................................................77
Install the Gloves ......................................................77
Assemble the Airflow Piping ......................................84
Install the Intake and Exhaust Filters .......................88
Stock and Inflate the Isolator ....................................92
Clean the Inside of the Isolator ..................................96
Start the Isolator .......................................................99
Prefilters .........................................................................99
Contents ix
7 Port Entry and Exit ............................................................101
Introduction .......................................................................101
Supplies .............................................................................101
Preparing, Opening, and Closing the Port ...........................102
Proper Placement of Materials.............................................107
8 Sterilizing Food and Supplies .............................................109
Introduction .......................................................................109
Sterility Indicators ..............................................................109
Assembling Supply Cylinders: Installing HEPA Filtration
Media .................................................................................112
Supplies .............................................................................112
Prepare the Cylinder...........................................................113
Fill the Cylinder .................................................................114
Water Sterilization ..............................................................122
Supplies ........................................................................125
Procedure .....................................................................126
9 Supply Cylinder Entry ........................................................129
Introduction .......................................................................129
Supplies .............................................................................130
Procedure ...........................................................................130
10 Isolator Maintenance ..........................................................139
Introduction .......................................................................139
Part 1: Cleaning and Maintenance......................................139
Part 2: Replacing Gloves on an Active Isolator .....................142
Procedure .....................................................................143
Part 3: Repairing Holes in Isolators ....................................147
Part 4: Working in the Isolator ............................................149
Sample and Tube Labeling Inside the Isolator ...............149
Weighing Mice...............................................................149
Options for Weighing Mice .......................................149
Use of the Pesola Scale for Weighing Mice
Aseptically ..............................................................150
11 Aseptic Mouse Transfer ......................................................155
Introduction .......................................................................155
x Contents
Part 1: Internal Transfer between isolators .........................155
Introduction ..................................................................155
Supplies ........................................................................156
Procedure .....................................................................156
Part 2: External Transfer of Mice ........................................157
Procedure .....................................................................157
12 Working with Germ-Free or Gnotobiotic Mice
in the Class II Biosafety Cabinet .........................................161
Introduction .......................................................................161
Supplies .............................................................................162
Procedure ...........................................................................162
Experimental Manipulations in the Biosafety Cabinet ........164
Introduction ..................................................................164
Procedures ....................................................................166
13 Shipping Mice .....................................................................171
Introduction .......................................................................171
Procedure ...........................................................................172
14 Rederivation .......................................................................181
Introduction .......................................................................181
Derivation by Cesarean Section ..........................................182
Derivation by Embryo Transfer ...........................................184
References ..........................................................................188
15 Microbiological Testing .......................................................189
Introduction .......................................................................189
Part 1: Screening for Bacterial Contamination ....................191
Supplies ........................................................................191
Sample Collection .........................................................191
Bacterial Culture and Slide Preparation ........................191
Gram Stain Procedure ..................................................195
Quantitative PCR ..........................................................196
Part 2: Examination of Gram-Stained Slides for the
Presence of Bacteria ...........................................................197
References ..........................................................................203
