Sorting of Bacteria

Kristi R. Harkins1

1 Iowa State University, Ames, Iowa
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.4
DOI:  10.1002/0471142956.cy1104s07
Online Posting Date:  May, 2001
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This unit provides detailed instructions for the sorting of organisms using the cell sorter. These techniques are very valuable for establishing pure cultures of organisms, for example those expressing GFP. The unit provides information on the culture, sorting, reculture, and verification of the sort purity and GFP expression. For those venturing into the area of bacterial sorting, this unit is a must.

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Table of Contents

  • Basic Protocol 1: Instrument Preparation for Bacteria Sorting
  • Basic Protocol 2: Bacterial Sorting for Enrichment or Cloning
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Instrument Preparation for Bacteria Sorting

  • 70% (v/v) ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A) filtered to remove particles >0.22 µm and autoclaved for 20 min
  • 0.5% (w/v) sodium hypochlorite
  • LB liquid medium (see recipe)
  • Cell sorter with 488‐nm excitation (15 mW)
  • Luer lock fittings: male and female luer‐to‐tubing adapters
  • Sterivex GP Filter (Millipore)
  • 10‐µm alignment beads
  • 2‐µm YG beads (Polysciences) diluted 1:3000 in filtered PBS
  • 0.5‐µm YG beads (Polysciences) diluted 1:5000 in filtered PBS
  • 12 × 75–mm tubes, sterile
  • 17 × 120–mm (15‐ml) conical tubes, sterile (Sort Collection Tubes)

Basic Protocol 2: Bacterial Sorting for Enrichment or Cloning

  • Log‐phase bacterial culture
  • 0.85% (w/v) NaCl (saline solution)
  • 70% (v/v) ethanol
  • LB liquid medium or 100 × 15–mm LB plates (see reciperecipes)
  • Cell sorter with jet‐in‐air or SortSense tip (Beckman Coulter), cloning device (e.g., Autoclone; Beckman Coulter), and air‐cooled argon laser (488 nm)
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Literature Cited

   Azuma, T., Harrison, G.I., and Demain, A.L. 1992. Isolation of gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system. Appl. Microbiol. Biotechnol. 38:173‐178.
   Beck, P. and Huber, R. 1997. Detection of cell viability in cultures of hyperthermophiles. FEMS Microbiol. Lett. 147:11‐14.
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single‐cell analyses. Microbiol. Rev. 60:641‐696.
   Francisco, J., Campbell, R., Iverson, B., and Georgiou, G. 1993. Production and fluorescence activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc. Natl. Acad. Sci. U.S.A. 90:10444‐10448.
   Mitchell, J.G., Weller, R., Beconi, M., Sell, J., and Holland, J. 1993. A practical optical trap for manipulating and isolating bacteria from complex microbial communities. Microb. Ecol. 25:113‐119.
   Nebe‐von Caron, G., Stephens, P., and Badley, R.A. 1996. Bacterial detection and differentiation by flow cytometry and fluorescent probes. In The Purdue Cytometry CD‐ROM Series, Vol. 2 (J.P. Robinson, ed.). Purdue University, West Lafayette, Ind.
   Nebe‐von Caron, G., Stephens, P., and Badley, R.A. 1998. Assessment of bacterial viability status by flow cytometry and single cell sorting. J. Appl. Microbiol. 84:988‐998.
   Nir, R., Yasraeli, Y., Lamed, R., and Sahar, E. 1990. Flow sorting of viable bacteria and yeasts according to β‐galactosidase activity. Appl. Environ. Microbiol. 56:3861‐3866.
   Porter, J., Deere, D., Pickup, R., and Edwards, C. 1996. Fluorescent probes and flow cytometry: New insights into environmental bacteriology. Cytometry 23:91‐96.
   Porter, J., Mobbs, K., Hart, C.A., Saunders, J.R., Pickup, R.W., and Edwards, C. 1997. Detection distribution and probable fate of Escherichia coli 0157 from asymptomatic cattle on a dairy farm. J. Appl. Microbiol. 33:297‐306.
   Ryan, C., Nguyen, B.T., and Sullivan, S.J. 1995. Rapid assay for mycobacterial growth and antibiotic susceptibility using gel microdrop encapsulation. J. Clin. Microbiol. 33:1720‐1726.
   Safarik, I., Safarikova, M., and Forsythe, S.J. 1995. The application of magnetic separations in applied microbiology. J. Appl. Bacteriol. 78:575‐585.
   Schmid, I., Nicholson, J.K.A., Giorgi, J.V., Janossy, G., Kunkl, A., Lopez, P.A., Perfetto, S., Seamer, L.C., and Dean, P.N. 1997. Biosafety guidelines for sorting of unfixed cells. Cytometry 28:99‐117.
   Tombolini, R., Unge, A., Davey, M.E., De Bruijn, F.J., and Jansson, J.K. 1997. Flow cytometric and microscopic analysis of GFP‐tagged Pseudomonas fluorescens bacteria. FEMS Microbiol. Ecol. 22:17‐28.
   United States Department of Health and Human Services (U.S. DHHS). 1993. National Institute of Health: Biosafety in Microbiological and Biomedical Laboratories, 3rd ed. Publication No. (CDC) 93‐8395, 993. U.S. Government Printing Office, Washington, D.C.
   Votyakova, T.V., Mukamolova, G.V., Shtein‐Margolina, V.A., Popov, V.I., Davey, H.M., Kell, D.B., and Kaprelyants, A.S. 1998. Research on the heterogeneity of Micrococcus luteus culture during an extended stationary phase: Subpopulation separation and characterization. Microbiology 67:85‐92.
   Wallner, G., Fuchs, B., Spring, S., Beisker, W., and Amann, R. 1997. Flow sorting of microorganisms for molecular analysis Appl. Environ. Microbiol. 63:4223‐4231.
Key Reference
   Davey and Kell, 1996. See above.
  An excellent overview of the problems and advantages associated with cytometric analysis of microorganisms; a section on cell sorting describes applications where bacterial sorting is of proven or probable benefit to researchers, and provides references.
Internet Resources
  Contains the material on flow cytometry and microbiology included in the Purdue Cytometry CD‐ROM Series Vol. 2, 1996.
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