Assessment of Enteral Bacteria

Alessandro Menozzi1, Maria Cristina Ossiprandi1

1 University of Parma, Parma, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 21.3
DOI:  10.1002/0471140856.tx2103s44
Online Posting Date:  May, 2010
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Abstract

The disruption of intestinal barrier leads to the penetration of noxious luminal compounds 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 sample, 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 aerobes 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. © 2010 by John Wiley & Sons, Inc.

Keywords: intestinal damage; bacteria; permeability

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessing Enteric Bacteria Number
  • Support Protocol 1: Preparation of TSA Plates for Aerobic Growth
  • Support Protocol 2: Preparation of Blood Agar Base Plates for Anaerobic Growth
  • Alternate Protocol 1: Using the Inclusion Method to Culture Anaerobic Bacteria
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assessing Enteric Bacteria Number

  Materials
  • Diethyl ether (Merck)
  • Rats (Wistar, males, 220 to 240 g body weight)
  • 40 mg/ml chlorhexidine gluconate (Hibiscrub)
  • 0.9% (w/v) NaCl solution, sterile
  • Sterile Ringer solution (Oxoid)
  • TSA culture plates ( protocol 2)
  • Blood agar base culture plates ( protocol 3)
  • Laminar vertical air flow hood (Bio‐Air)
  • Foam board with pins
  • Hair clipper (Oster)
  • Sterile gauzes
  • Sterile surgical gloves
  • Sterile surgical towels
  • Sterile surgical instruments including:
    • Blunt‐end scissors
    • Sharp‐end scissors
    • Surgical forceps
  • 100‐ml beakers
  • 90‐mm petri dishes, sterile
  • Stomacher bags, sterile (Laboindustria)
  • Stomacher (PBI)
  • 15‐ml polypropylene round‐bottom tubes (Becton Dickinson), sterile
  • 0.1‐, 1‐, and 10‐ml pipets (Sarstedt), sterile
  • Pipet aid (Drummond)
  • Micropipettors (Gilson)
  • Pipet tips (Sarstedt)
  • Analytical scale (Acculab)
  • Vortex mixer (Falc Instrument)
  • Sterile spreaders (Laboindustria)
  • Incubator 37 ± 1°C (Instruments)
  • Anaerobic jars (Oxoid)
  • Anaerobic sachets (Oxoid)
  • Colony counter (PBI)

Support Protocol 1: Preparation of TSA Plates for Aerobic Growth

  Materials
  • Tryptone Soya Agar, TSA (Oxoid)
  • Plate Count Agar, PCA (Oxoid), optional
  • Distilled water
  • Appropriately sized flasks
  • Analytical scale (Acculab)
  • Magnetic heated stirrer (PBI)
  • Magnetic stirrer (PBI)
  • Laboratory autoclave (Fedegari)
  • Autoclave gloves (Clavies)
  • 50°C water bath
  • Laminar air horizontal flow (Gelman Instruments)
  • Petri dishes (Sarstedt)

Support Protocol 2: Preparation of Blood Agar Base Plates for Anaerobic Growth

  Materials
  • Blood Agar Base powder (Oxoid)
  • Schaedler Agar ready prepared plates (Oxoid), optional
  • Distilled water
  • Sterile defibrinated blood (Oxoid)
  • Analytical scale (Acculab)
  • Appropriately sized flasks
  • Magnetic heated stirrer (PBI)
  • Magnetic stirrer (PBI)
  • Laboratory autoclave (Fedegari)
  • Autoclave gloves (Clavies)
  • 50°C water bath
  • Laminar air horizontal flow hood (Gelman Instruments)
  • 1‐ and 10‐ml pipets (Sarstedt), sterile
  • Pipet aid (Drummond)
  • Petri dishes (Sarstedt)

Alternate Protocol 1: Using the Inclusion Method to Culture Anaerobic Bacteria

  • Diluted samples of intestinal homogenate ( protocol 1, steps 13)
  • Blood agar base ( protocol 3, steps 1 through 11), 45°C
  • Vortex
  • Petri dishes
NOTE: Maintain sterility of these cultures by working under a laminar horizontal airflow hood.
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Figures

  •   FigureFigure 21.3.1 Assessing colony numbers by means of Colony Counter. The plate is put upside‐ down on the illuminated glass and the colonies are tallied directly on the back of the petri dish using a marker pen. The counter records the number of pressures and shows it on the display.
  •   FigureFigure 21.3.2 Comparison between a petri dish (TSA) with optimal culture of enteric aerobic bacteria, ready for the count of the colonies (A), and a bad culture (B) with typical colony growth on the dish perimeter, indicative of environmental contamination. The latter dish has to be discarded.

Videos

Literature Cited

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   Berg, R.D. 1995. Bacterial translocation from the gastrointestinal tract. Trends Microbiol. 3:149‐154.
   Berg, R.D. and Garlington, A.W. 1979. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in gnotobiotic mouse model. Infect. Immunol. 23:403‐411.
   Berg, R.D., Wommack, E., and Deitch, E.A. 1988. Immunosuppression and intestinal bacterial overgrowth synergistically promote bacterial translocation. Arch. Surg. 123:1359‐1364.
   Bjarnason, I., Williams, P., Smethurst, P., Peters, T.J., and Levi, A.J. 1986. The effect of NSAIDs and prostaglandins on the permeability of the human intestine. Gut 27:1292‐1297.
   Bjarnason, I., Macpherson, A., and Hollander, D. 1995. Intestinal permeability: An overview. Gastroenterology 108:1566‐1581.
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   João, S.A., Alencar, S.S., Medeiros, A.C., Diniz. S.O., Cardoso, V.N., and Brandt, C.T. 2004. Translocation of 99mTc labelled bacteria after intestinal ischemia and reperfusion. Acta Cir. Bras. 19:328‐333.
   Guo, W., Andersson, R., Willén, R., Ljungh, A., Wang, X., Liu, X., and Bengmark, S. 1994. Bacterial translocation after intraperitoneal implantation of rubber fragments in the splenectomized rat. J. Surg. Res. 57:408‐415.
   Madara, J.L. 1990. Warner‐Lambert/Parke‐Davis Award lecture. Pathobiology of the intestinal epithelial barrier. Am. J. Pathol. 137:1273‐1281.
   Mahmud, T., Rafi, S.S., Scott, D.L., Wrigglesworth, J.M., and Bjarnason, I. 1996. Nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation. Arthritis Reum. 39:1998‐2003.
   Matthews, J.B., Smith, J.A., Tally, K.J., Menconi, M.J., Nguyen, H., and Fink, M.P. 1994. Chemical hypoxia increases junctional permeability and activates electrogenic ion transport in human intestinal epithelium monolayers. Surgery 116:157‐158.
   Menozzi, A., Pozzoli, C., Giovannini, E., Solenghi, E., Grandi, D., Bonardi, S., Bertini, S., Vasina, V., and Coruzzi, G. 2006. Intestinal effects of nonselective and selective cyclooxygenase inhibitors in the rat. Eur. J. Pharmacol. 552:143‐150.
   O'Boyle, C.J., MacFie, J., Mitchell, C.J., Johnstone, D., Sagar, P.M., and Sedman, P.C. 1998. Microbiology of bacterial translocation in humans. Gut. 42:29‐35.
   Reuter, B.K., Davies, N.M., and Wallace, J.L. 1997. Nonsteroidal anti‐inflammatory drug enteropathy in rats: Role of permeability, bacteria and enterohepatic circulation. Gastroenterology 112:109‐117.
   Reynolds, J.V., Murchan, P., Leonard, N., Gough, D.B., Clarke, P., Keane, F.B., and Tanner, W.A. 1995. High‐dose interleukin 2 promotes bacterial translocation from the gut. Br. J. Cancer. 72:634‐636.
   Robert, A. and Asano, T. 1997. Resistance of germ‐free rats to indomethacin‐induced intestinal lesions. Prostaglandins 14:333‐341.
   Sedman, P.C., MacFie, J., Sagar, P., Mitchell, C.J., May, J., Mancey‐Jones, B., and Johnstone, D. 1994. The prevalence of gut translocation in humans. Gastroenterology 107:643‐649.
   Simon, G.L. and Gorbach, S.L. 1986. The human intestinal microflora. Dig. Dis. Sci. 31:147S‐162S.
   Somasundaram, S., Hyallar, J., Rafi, S., Wrigglesworth, J.M., Macpherson, A.J., and Bjarnason, I. 1995. The biochemical basis of NSAID‐induced damage to the gastrointestinal tract: A review and a hypothesis. Scand. J. Gastroenterol. 30:289‐299.
   Steinberg, S.M. 2003. Bacterial translocation: What it is and what it is not. Am. J. Surg. 186:301‐305.
   Taurog, J.D., Richardson, J.A., Croft, J.T., Simmons, W.A., Zhou, M., Fernández‐Sueiro, J.L., Balish, E., and Hammer, R.E. 1994. The germfree state prevents development of gut and joint inflammatory disease in HLA‐B27 transgenic rats. J. Exp. Med. 180:2359‐2364.
   Yamada, T., Deitch, E., Specian, R.D., Perry, M.A., Sartor, R.B., and Grisham, M.B. 1993. Mechanisms of acute and chronic intestinal inflammation induced by indomethacin. Inflammation 17:641‐662.
   Zanoni, F.L., Benabou, S., Greco, K.V., Moreno, A.C., Costa Cruz, J.W., Filgueira, F.P., Martinez, M.B., de Figuereido, L.F., Rocha e Silva, M., and Sannomiya, P. 2009. Mesenteric microcirculatory dysfunctions and translocation of indigenous bacteria in a rat model of strangulated small bowel obstruction. Clinics 64:911‐919.
Key Reference
   Balzan et al., 2007. See above
  This paper provides an excellent overview of pathogenic mechanisms of bacterial translocation in health and disease, as well as the methods used for its assessment.
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