Flow Cytometric Detection of Pathogenic E. coli in Food

Richard B. Raybourne1

1 U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition Laurel, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.6
DOI:  10.1002/0471142956.cy1106s08
Online Posting Date:  May, 2001
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Abstract

E. coli O157:H7 is one of the more important food pathogens, andrapid, quantitative methods to evaluate foods for the presence of this pathogen are needed. This unit provides exactly that: a very much simplified flow cytometric assay for detection of E. coli O157:H7 in a well established vehicle of infection, ground beef. The method uses commercially available FITC‐conjugated specific antibody to this bacterial serotype. Sample preparation and bacterial enrichment procedures are described. Direct and indirect approaches for quantification of the number of bacteria are given. A key feature of the assay is the reduction in time compared with plate‐counting methods; the tradeoff is a slight reduction in sensitivity. Particularly useful is the simultaneous inclusion of a spiked sample to ensure a positive control. In addition, the unit provides hints on sorting the organisms if desired.

     
 
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Table of Contents

  • Basic Protocol 1: Calibration and Standardization of Flow Cytometry with Conventional Culture Methods to Determine Lower Limit of Detection and to Detect Pathogenic E. coli in a Food Matrix
  • Alternate Protocol 1: Direct Enumeration of E. coli O157:H7 Using Metered Sample Flow
  • Support Protocol 1: Optimization of the Flow Cytometer for Analysis of Bacteria
  • Support Protocol 2: Sorting of Fluorescent E. coli O157:H7
  • Support Protocol 3: Selection of Streptomycin‐Resistant E. coli O157:H7
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Calibration and Standardization of Flow Cytometry with Conventional Culture Methods to Determine Lower Limit of Detection and to Detect Pathogenic E. coli in a Food Matrix

  Materials
  • Ground beef sample
  • E. coli O157:H7, streptomycin resistant (ATCC #35150; see protocol 5)
  • Enrichment culture (EC) broth (Difco) with 20 µg/ml novobiocin (Difco)
  • Luria‐Bertani broth (Difco)
  • Dulbecco's PBS (Life Technologies)
  • MacConkey sorbitol agar (Difco) containing dihydrostreptomycin or other appropriate antibiotic (Life Technologies)
  • 10 mg/ml trypsin in PBS
  • 0.5% (v/v) Triton X‐100
  • FITC‐labeled rabbit anti–E. coli O157:H7 (Kirkegaard & Perry)
  • ∼106 bead/ml suspension of 10‐µm polystyrene fluorescent beads (Beckman Coulter)
  • Stomacher Model 400 laboratory blender (Seward)
  • 12 × 75–mm polypropylene tubes (Falcon)
  • 50°C water bath
  • 10‐ml plastic syringes with 5‐µm syringe‐tip filters (nylon Swinnex‐type; Fisher)
  • Flow cytometer with 480 to 490 nm excitation beam and log forward light scatter amplification, optimized for analysis of bacteria (see protocol 3)

Alternate Protocol 1: Direct Enumeration of E. coli O157:H7 Using Metered Sample Flow

  • Flow cytometer with metered sample delivery; e.g., Bryte HS (Bio‐Rad)

Support Protocol 1: Optimization of the Flow Cytometer for Analysis of Bacteria

  • Standard laboratory strain (i.e., non‐O157:H7) of E. coli, e.g., K12 (ATCC #10798)
  • Sheath fluid: distilled water or PBS prefiltered through a 0.1‐ to 0.2‐µm‐pore‐size filter

Support Protocol 2: Sorting of Fluorescent E. coli O157:H7

  • Selective and nonselective agar: LB agar (Difco) with and without 500 µg/ml dihydrostreptomycin, respectively
  • Flow cytometer capable of cell sorting
  • Microplate deposition device: e.g., Autoclone (Beckman Coulter)
  • Sterile 96‐well plates (optional)

Support Protocol 3: Selection of Streptomycin‐Resistant E. coli O157:H7

  • Luria‐Bertani (LB) broth (Difco) with 0, 50, and 500 µg/ml dihydrostreptomycin (Life Technologies)
  • Luria‐Bertani (LB) agar plates (Difco) with 50 and 500 µg/ml dihydrostreptomycin
  • Glycerol, sterile
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Figures

Videos

Literature Cited

Literature Cited
   Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., and Stahl, D.A. 1990. Combination of 16S rRNA‐targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919‐1925.
   CAST Task Force. 1994. Foodborne Pathogens: Risks and Consequences. Council for Agricultural Science and Technology. Ames, Iowa.
   Donnelly, C.W. and Baigent, G.J. 1986. Method for flow cytometric detection of Listeria monocytogenes in milk. Appl. Environ. Microbiol. 52:689‐695.
   Food and Drug Administration (FDA) 1995. Bacteriological Analytical Manual, 8th ed. AOAC International, Gaithersburg, Md.
   Fung, D.Y.C. 1994. Rapid methods and automation in food microbiology: A review. Food Rev. Int. 10:357‐375.
   McClelland, R.G. and Pinder, A.C. 1994a. Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies. Appl. Environ. Microbiol. 60:4255‐4262.
   McClelland, R.G. and Pinder, A.C. 1994b. Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry. J. Appl. Bacteriol. 77:440‐447.
   Raybourne, R.B. 1997. Flow cytometry in food microbiology: Detection of Escherichia coli O157:H7. In Food Microbiological Analysis New Technologies. (M.L. Tortorello and S.M. Gendel, eds.) pp. 57‐68, Marcel Dekker, New York.
   Steen, H.B. 1990. Light scattering measurement in an arc lamp‐based flow cytometer. Cytometry 11:223‐230.
   Tortorello, M.L., Reineke, K.F., Stewart, D.S., and Raybourne, R.B. 1998. Comparison of methods for determining the presence of Escherichia coli O157:H7 in apple juice. J. Food Prot. 61:1425‐1430.
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