Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration

Kristi R. Harkins1, Kelley Harrigan1

1 Advanced Analytical Technologies, Inc., Ames, Iowa
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.17
DOI:  10.1002/0471142956.cy1117s29
Online Posting Date:  September, 2004
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Abstract

Traditional uses of flow cytometry have been for research and diagnostic purposes, on mammalian cells. This unit focuses on using a flow cytometer for enumerating specific bacteria and parasites. Several different labeling methods are discussed, as well as how to set up a cytometer for detecting and enumerating bacteria. Labeling methods include direct detection with a primary fluorochrome‐conjugated antibody and indirect labeling with an unconjugated primary and a fluorochrome‐conjugated secondary antibody, as well as labeling with rRNA sequence‐specific peptide nucleic‐acid probes end‐labeled with a fluorochrome. Data and methods throughout this unit focus on detection of Salmonella in clean matrices; however, pertinent information is also provided on using these methods to label other pathogens. The Commentary covers critical aspects of the protocols, and also includes information and suggestions on the application of these methods for testing in the food and pharmaceutical industries, as well as in environmental water testing.

Keywords: Salmonella; Antibody; rRNA; FISH; Enumeration; Detection; Food; Water

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Cytometer for Detection of Microorganisms
  • Basic Protocol 2: Direct Labeling of a Pathogen (Dilution)
  • Support Protocol 1: Correlation of Flow Cytometric Data with Traditional Plate‐Count Enumeration
  • Alternate Protocol 1: Direct Antibody Labeling (Cell Pellet)
  • Alternate Protocol 2: Indirect Pathogen Labeling
  • Basic Protocol 3: In Situ Hybridization of rRNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Cytometer for Detection of Microorganisms

  Materials
  • Sheath fluid: 25 µM cetyltrimethylammonium bromide (CTAB) in deionized water (or prepared as recommended by flow cytometer manufacturer), filtered through 0.22‐µm filter
  • 2.5‐µm Deep Red beads (Molecular Probes): 0.04%, 0.2%, and 0.8% intensities
  • Flow cytometer with 635‐nm excitation and filters for detection of far‐red fluorescence at 700 nm
  • Luer‐lock fitting (male and female luer‐to‐tubing adapters)
  • Sterivex GP filter (Millipore)

Basic Protocol 2: Direct Labeling of a Pathogen (Dilution)

  Materials
  • Goat anti‐Salmonella antibody (Kirkegaard & Perry)
  • Alexa Fluor 647 (Molecular Probes) or Cy5 (Amersham Biotech) dye conjugation kit
  • Glycerol
  • PBS‐BSA: 0.05% (w/v) bovine serum albumin in PBS (APPENDIX appendix 2A), filtered through 0.22‐µm filter
  • Overnight culture ( appendix 3F) of Salmonella typhimurium (ATCC #14028)
  • Tryptic soy broth (e.g., Difco)
  • Bacterial sample for analysis
  • Millex‐GV 0.2‐µm syringe filter unit (Millipore)
  • Flow cytometer with 635‐nm excitation and filters for collection of far‐red fluorescence at 700 nm
  • 12 × 75–mm sterile test tubes

Support Protocol 1: Correlation of Flow Cytometric Data with Traditional Plate‐Count Enumeration

  • Solid medium ( appendix 3E) appropriate for bacteria of interest (use selective media for non‐pure culture or nonselective media for pure cultures; for pure Salmonella culture use nonselective tryptic soy broth)
  • Petri dishes, 100 × 15 mm
  • Additional reagents and equipment for plating bacteria ( appendix 3G)

Alternate Protocol 1: Direct Antibody Labeling (Cell Pellet)

  • Microcentrifuge capable of 20,800 × g
  • 2‐ml microcentrifuge tubes, sterile

Alternate Protocol 2: Indirect Pathogen Labeling

  • Goat anti‐Salmonella primary antibody (Kirkegaard & Perry), unconjugated
  • Rabbit anti‐goat (or any other anti‐goat) secondary antibody labeled with Cy5 (Jackson ImmunoResearch)
  • Microcentrifuge capable of 20,800 × g
  • 2‐ml microcentrifuge tubes, sterile

Basic Protocol 3: In Situ Hybridization of rRNA

  Materials
  • Lyophilized Cy5‐PNA Sal probe (Applied Biosystems; store at −20°C; see probe sequence in Table 11.17.2)
  • Dimethylformamide (DMF)
  • 16% (w/v) formaldehyde (methanol‐free, Polysciences)
  • Log‐phase culture ( appendix 3F) of Salmonella typhimurium (ATCC #14028)
  • PBS‐BSA: 0.05% (w/v) bovine serum albumin in PBS ( appendix 2A), filtered through 0.22‐µm filter
  • Hybridization buffer (see recipe), optimized for stringency (see Critical Parameters)
  • Wash buffer (see recipe), optimized for stringency (see Critical Parameters)
  • Temperature‐controlled heat block
  • Microcentrifuge capable of 20,800 × g
  • Flow cytometer with 635‐nm excitation and filters to collect emission at 700 nm
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Figures

Videos

Literature Cited

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   Hughes, E.E., Matthews‐Greer, J.M., and Gilleland, H.E.J. 1996. Analysis by flow cytometry of surface‐exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Can. J. Microbiol. 42:859‐862.
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   Perry‐O'Keefe, H., Rigby, S., Oliveira, K., Sorensen, D., Stender, H., Coull, J., and Hyldig‐Nielsen, J.J. 2001. Identification of indicator microorganisms using a standardized PNA FISH method. J. Microbiol. Methods 47:281‐292.
   Srikumar, R., Chin, A.C., Vachon, V., Richardson, C.D., Ratcliffe, M.J. Saarinen, L., Keyhty, H., and Coulton, J.W. 1992. Monoclonal antibodies specific to porin of Haemophilus influenzae type b: Localization of the cognate epitopes and test for their biological activities. Mol. Microbiol. 6:665‐676.
   Stender, H., Broomer, A., Oliveira, K., Perry‐O'Keefe, H., Hyldig‐Nielsen, J.J., Sage, A., Young, B., and Coull, J. 2000. Rapid detection, identification and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes. J. Microbiol. Methods 42:245‐253.
   Stender, H., Broomer, A.J., Oliveira, K., Perry‐O'Keefe, H., Hyldig‐Nielsen, J.J., Sage, A., and Coull, J. 2001. Rapid detection, identification and enumeration of E. coli cells in municipal water by chemiluminescent in situ hybridization. Appl. Environ. Microbiol. 67:142‐147.
   Stender, H., Fiandaca, M., Hyldig‐Nielsen, J.J., and Coull, J. 2002. PNA for rapid microbiology. J. Microbiol. Methods 48:1‐17.
   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.
   Vesey, G., Slade, J.S., Byrne, M., Shepherd, K., Dennis, P.J., and Fricker, C.R. 1993. Routine monitoring of Cryptosporidium oocyst in water using flow cytometry. J. Appl. Bacteriol. 75:87‐90.
   Vesey, G., Narai, J., Ashbolt, N., Williams, K.L., and Veal, D.A. 1994. Detection of specific microorganisms in environmental samples by flow cytometry. In Methods in Cell Biology, Vol. 42 (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp. 482‐522. Academic Press, New York, N.Y.
   Vesey, G., Ashbolt, N., Fricker, E.J., Deere, D., Williams, K.L., Veal, D.A., and Dorsch, M. 1998. The use of a ribosomal RNA targeted oligonucleotide prove for fluorescent labeling of viable Cryptosporidium parvum oocyst. J. Appl. Microbiol. 85:429‐440.
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   Zoetendal, E.G., Ben‐Amor, K., Harmsen, H.J.M, Schut, F., Akkermans, A.D.L., and de Vos, W.M. 2002. Quantification of uncultured Ruminococcus obeum‐like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA‐targeted probes. Appl. Environ. Microbiol. 68:4225‐4232.
Internet Resources
  http://www.orcbs.msu.edu/biological/BMBL/BMBL‐1.htm
  United States Department of Health and Human Services, National Institute of Health: Biosafety in Microbiological and Biomedical Laboratories, 3rd ed. Publication No. (CDC) 93‐8395, 993.
   http://www.epa.gov/nerlcwww/
  This EPA method 1623 can be found here.
  http://www.epa.gov/nerlcwww/1623ap01.pdf
  EPA method for concentrating and enumerating Cryptosporidium oocysts.
  http://www.psb.ugent.be/rRNA/
  Searchable rRNA sequence data base for a variety of bacteria types.
  http://www.cfsan.fda.gov/∼ebam/bam-a1.html
  Appendix of rapid methods for detecting food pathogens from the on‐line Bacteriological Analytical Manual.
  http://www.linscottsdirectory.com
  A service‐for‐fee site that allows the user to search for vendors that produce antibodies to pathogens.
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