UNIT 21.3 Assessment of Enteral Bacteria Alessandro Menozzi 1 and Maria Cristina Ossiprandi 1 1 University of Parma, Parma, Italy ABSTRACT The disruption of intestinal barrier leads to the penetration of noxious luminal com- pounds into the gut wall, causing further damage. This unit describes the assessment of enteric bacteria translocation into the intestinal wall of rats, an established method for the evaluation of bowel damage to the mucosal epithelial barrier. The Basic Protocol provided in the present unit describes collection and preparation of small intestine sam- ple, performing of sample serial dilutions for bacterial culture, performing of the culture of aerobic and anaerobic bacteria on petri dishes, incubation of the cultured plates, and counting of bacterial colonies. The Support Protocols describes the procedures for the preparation of petri dishes for the culture, using different employable media for aer- obes or anaerobes. The Alternate Protocol describes the use of the “inclusion method,” suitable for the culture of anaerobic bacteria. Curr. Protoc. Toxicol. 44:21.3.1-21.3.11. C  2010 by John Wiley & Sons, Inc. Keywords: intestinal damage  bacteria  permeability INTRODUCTION Among the several functions of intestinal epithelium, the ability to work as an effective shield against lumen noxious constituents is remarkable, considering that this barrier is made of a single lining of cells. The protective layers, which concur to hinder the penetration of potentially harmful compounds, include an extrinsic barrier that is made of a water lining and a mucous film, also known as glycocalix, mostly made of glycolipids and glycoproteins, and an intrinsic barrier constituted by enterocytes and paracellular spaces between these cells (Madara, 1990). At the apex of the intercellular space are located the so-called tight junctions, the most important structure of the intestinal barrier, which hold epithelial cells close together and block the permeation of microorganisms and large compounds such as hydrophilic macromolecules (proteins, LPS, or peptidoglycan, for instance; Balzan et al., 2007). The efficacy of this sophisticated barrier is therefore dependent on the integrity of the enterocyte brush-border membranes and of the tight junctions, besides the ability to produce the right array of epithelial secretory products. Many toxic substances and intestinal diseases are able to disrupt the enteric epithelial barrier. Intestinal damage is often characterized by lesions ranging from microscopic ero- sions of the mucosa to large macroscopic ulcers or even perforations. When the anatom- ical structure of the intrinsic barrier is severely altered and the enterocyte monolayer becomes discontinuous, there is obviously no protection from the passage of macro- molecules from the lumen to inner tissues. In several cases, however, intestinal damage can be more subtle and devious and, even if the wholeness of the epithelium seems to be preserved, tight junctions can become dysfunctional and large molecules are able to pass through and to trigger further damage. Increased gut permeability is thought to be relevant in the pathogenesis of many intestinal diseases like food allergies, inflammatory bowel disease (IBD), autoimmune disorders, celiac sprue, and the damage induced by radiations, toxins, and drugs (Bjarnason et al., 1995). There is evidence, as a matter of fact, that one of the crucial initiating events of Current Protocols in Toxicology 21.3.1-21.3.11, May 2010 Published online May 2010 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471140856.tx2103s44 Copyright C  2010 John Wiley & Sons, Inc. Gastrointestinal Toxicology 21.3.1 Supplement 44