Gnotobiotic mouse technology : an illustrated guide PDF
Preview Gnotobiotic mouse technology : an illustrated guide
GGnnoottoobbiioottiicc 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. 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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
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