• Abstract
• Table of Contents
• Materials
• Figures
• Literature Cited

Abstract

The normal gut microbiota has evoked many investigators' interest over the years and the pioneering work of James Reyniers in the 1920s generated the first germ?free guinea pigs. Comparing the physiology between germ?free and conventionally raised animals has provided invaluable insights on how the gut microbiota affect host biology. Today we know that the gut microbiota modulate the immune system, epithelial cell proliferation, intestinal angiogenesis, hormone production, energy absorption, and behavior. Furthermore, recent data have demonstrated that obesity is associated with an altered gut microbiota, and a direct role for the microbiota in disease development was demonstrated by the use of germ?free mice. Here we are presenting protocols for maintaining and generating germ?free mice. Curr. Protoc. Mouse Biol. 2:307?316 © 2012 by John Wiley & Sons, Inc.

Keywords: gut microbiota; gnotobiotic mouse models; hysterectomy

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Table of Contents

• Introduction
• Basic Protocol 1: Maintaining Germ‐Free Mice: Practical Considerations for Housing and Equipment Sterility
• Basic Protocol 2: Assessing Germ‐Free Status by Fecal Bacteria Analysis
• Alternate Protocol 1: Assessing Germ‐Free Status by Molecular Methods
• Basic Protocol 3: Rederivation of Mice
• Commentary
• Literature Cited
• Figures

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Materials

GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures

• Figure 1. Photos of 12‐week‐old germ‐free (left) and conventionally raised (right) Swiss Webster females.* denotes the cecum.

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• Figure 2. (A ) Isolator for housing germ‐free mice. (B ) Sterilizing cylinder.

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• Figure 3. Preparing food, bedding, and water for sterilization in cylinders. Individual cylinders should be used for each class of product and a Steris dart included in each cylinder to verify successful sterilization. Note: A = Mylar film over container to seal contents before loading into autoclave; B/C = single layer of Mylar tape (B) used to secure Mylar film (A), followed by three layers of vinyl tape (C) to ensure seal integrity. Also note that sterilization cylinders have been covered by filter paper ahead of time; this paper should suffice for at least 1 year, and is necessary to maintain sterility of contents, while permitting pressurized steam penetration. Contents not drawn to scale.

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• Figure 4. General process for importing autoclaved supplies into sterile isolator. (A ) [1] Autoclaved, cooled, sealed sterilization cylinder containing Steris dart (note indicator color change from yellow to black) and food/bedding/water; [2] transfer sleeve (note nipples—used for fumigation); [3] transfer port of isolator, still sealed on both outer and inner ends. (B ) [4] Sealed sterilization cylinder is attached to transfer sleeve and [5] sealed with three layers of vinyl tape (denoted by red stripe). (C ) [6] Outer end cover of isolator is removed and [7] connected to other end of transfer sleeve; [8] connection between transfer sleeve and transfer port also sealed with three layers of vinyl tape. (D ) [9] Fumigate interior of sleeve with Clidox (through sleeve nipples) and allow to stand for 1 hr. (E ) [10] Once fumigation is complete, remove inner isolator transfer port cover and [11] use sterile forceps to reach through the transfer port and sleeve to puncture the mylar film cover of the sterilization cylinder; [12] remove the autoclaved supplies from the cylinder (if desired, any contents within the isolator may be exported into the cylinder at this stage). (F ) [13] Reconnect the inner transfer port cap. (G ) [14] Disconnect the transfer sleeve from the isolator, [15] reconnect the outer transfer port cap (sterilize the transfer port with Clidox via outer cap nipples; outer cap nipples not shown in illustrations), and [16] disconnect the sterilization cylinder from transfer sleeve. Items not drawn to scale. NOTE: Outer and inner caps are secured using 18‐in rubber bands.

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