Cell Affinity Chromatography for a Marine Nitrifying Bacterium

  • U. Heyman
  • B. Heyman
  • Bess B. Ward
Part of the Lecture Notes on Coastal and Estuarine Studies book series (COASTAL, volume 25)


Difficulty in the identification and separation of species of bacteria remains a major obstacle to the understanding of microbial processes in aquatic environments. A routine, widely applicable method for obtaining total cell counts by means of acridine orange or other DNA-staining fluorochromes has been available for several years (Hobbie et al., 1977; Porter and Feig, 1980; Paul, 1982) and recently the use of immunofluorescent staining has made counts on separate species possible (Ward and Perry, 1980; Campbell et al., 1983; Dahle and Laake, 1982: Ward and Carlucci, 1985). Species specific antibodies can also be used in radioimmunoassay to enumerate bacteria (Benbough and Martin, 1976). Although these methods give reasonable estimates of abundance of cells of a particular strain, methods used to estimate metabolic activity of cells of individual strains are still inadequate. Microautoradiography (Fliermans and Schmidt, 1975: Ward, 1984) and ETS-activity (Baker and Mills, 1982) in combination with immunofluorescence have been applied with some success.


Acridine Orange Sepharose Bead Cell Mixture Preserve Cell Phenyltetrazolium Chloride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Baker, K. H. and A.L. Mills. 1982. Determination of the number of respiring Thiobacillus ferrooxidans cells in water samples by using combined fluorescent antibody-2-(p-iodophenyl) -3-(p-nitrophenyl)-5- phenyltetrazolium chloride staining. Appl. Environ. Microbiol. 43: 338–344.PubMedGoogle Scholar
  2. Benbough, J. E. and K.L. Martin. 1976. An indirect radiolabeled antibody staining technique for the rapid detection and identification of bacteria. Jour. Appl. Bact. 41: 47–58.CrossRefGoogle Scholar
  3. Carlucci, A.F. and D. Pramer. 1957. Factors influencing the plate method for determining abundance of bacteria in sea water. Proc. Soc. Exp. Biol. Med. 96: 392–394.PubMedGoogle Scholar
  4. Campbell, L., E.J. Carpenter and V.J. Iacono. 1983. Identification and enumeration of marine chroococcoid cyanobacteria by immunofluorescence. Appl. Environ. Microbiol. 46: 553–559.PubMedGoogle Scholar
  5. Chess, L., R.P. MacDermott and S. F. Schlossman. 1974. Immunologic functions of isolated human lymphocyte subpopulations. I. Quantitative isolation of human T and B cells and response to mitogens. J. Immunol. 113: 1113–1121.PubMedGoogle Scholar
  6. Dahle, A.B. and M. Laske. 1982. Diversity dynamics of marine bacteria studied by immunofluorescent staining on membrane filters. Appl. Environ. Microbiol. 43: 169–176.PubMedGoogle Scholar
  7. Davis, B.D., R. Dulbecco, H.N. Eisen, H.S. Ginsberg and W.B. Wood. 1973. Microbiology, 2nd ed. Harper and Row Publishers, Inc., Hagerstown, MD.Google Scholar
  8. Duarte, A.J.S., C.B. Carpenter and T.B. Strom. 1982. Expression of T cell differentiation antigens and Ia on rat cytotoxic T lymphocytes. Jour. Immunol. 128: 580Google Scholar
  9. Fliermans, C.B. and E.L. Schmidt. 1975. Autoradiography and immunofluorescence combined for autecological study of single cell activity with Citrobacter as a model system. Appl. Microbiol. 30: 676–684.PubMedGoogle Scholar
  10. Hanson, R.S. and J.A. Phillips. 1981. Chemical composition. IN: Manual of Methods for General Bacteriology. P. Gerhardt, (editor in chief). Amer. Soc. Microbiol., Washington, D.C. pp. 328–364.Google Scholar
  11. Hobbie, J.E., R.J. Daley and S. Jasper. 1977. Use of Nuclepore filters for counting bacteria by fluorescence microscopy, Appli. Environ. Microbiol. 33: 1225–1228.Google Scholar
  12. Hutchinson, G.E. 1967. A Treatise on Limnology. Vol. 2. John Wiley & Sons, Inc.Google Scholar
  13. Manderino, G.L., G.T. Gooch and A.B. Stavitsky. 1978. Preparation, characterization and functions of rabbit lymph node cell populations. I. Preparation of KLH primed T and B memory cells with anti-Fab’ affinity columns. Cell. Immunol., 41: 264–275.PubMedCrossRefGoogle Scholar
  14. Miller, T.J. and H.O Stone. 1978. The rapid isolation of ribonuclease-free immunoglobulin G by protein A-sepharose affinity chromatography. J. Immunol. Methods. 24: 111–125.PubMedCrossRefGoogle Scholar
  15. Paul, J.H. 1982. Use of Hoechst dyes 33258 and 33342 for enumeration of attached and planktonic bacteria. Appl. Environ. Microbiol. 43: 939–944.PubMedGoogle Scholar
  16. Porter, K.G. and Y.S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943–948.CrossRefGoogle Scholar
  17. Pharmacia Fine Chemicals AB. 1980. Cell Affinity Chromatography: Principles and Methods. Pharmacia Fine Chemicals, Uppsala, Sweden.Google Scholar
  18. Rouse, H. 1938. Fluid mechanics for hydraulic engineers. Dover Publications, Inc. New York.Google Scholar
  19. Sandstrom, G. and H.W. Watz. 1984. The duct ELISA: A new technique to identify low numbers of Fransiscella tularensis. FOA Report C 40202–133. National Defense Research Institute, S90182 Umea, Sweden.Google Scholar
  20. Ward, B.B. 1982. Oceanic distribution of ammonium-oxidizing bacteria determined by immunofluorescent assay. Jour. Mar. Res. 40: 1155–1172.Google Scholar
  21. Ward, B.B. 1984. Combined autoradiography and immunofluorescence for estimation of single cell activity by ammonium-oxidizing bacteria. Limnol. Oceanogr. 29: 402–410.CrossRefGoogle Scholar
  22. Ward, B.B. and A.F. Carlucci. 1985. Marine ammonia- and nitrite-oxidizing bacteria: Serological diversity determined by immunofluorescence in culture and in the environment. Appl. Environ. Microbiol. 50: 194–201.PubMedGoogle Scholar
  23. Ward, B.B. and M.J. Perry. 1980. Immunofluorescent assay for the marine ammonium-oxidizing bacterium Nitrosococcus oceanus. Appl. Environ. Microbiol. 39: 913–918.PubMedGoogle Scholar
  24. Wigzell, H. 1976. Specific affinity fractionation of lymphocytes using glass or plastic bead columns. Scand. Jour. Immunol. 5(suppl. 5): 23–30.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag New York, Inc. 1988

Authors and Affiliations

  • U. Heyman
    • 1
  • B. Heyman
    • 2
  • Bess B. Ward
    • 3
  1. 1.Institute of LimnologyUniversity of UppsalaUppsalaSweden
  2. 2.Department of ImmunologyUppsala University Biomedical CenterUppsalaSweden
  3. 3.Institute of Marine Resources Scripps Institution of OceanographyUniversity of California, San DiegoLa JollaUSA

Personalised recommendations