Microbial Ecology

, Volume 8, Issue 4, pp 313–323 | Cite as

Survival and viability of nonculturableEscherichia coli andVibrio cholerae in the estuarine and marine environment

  • Huai -Shu Xu
  • N. Roberts
  • F. L. Singleton
  • R. W. Attwell
  • D. J. Grimes
  • R. R. Colwell


Plating methods for estimating survival of indicator organisms, such asEscherichia coli, and water-borne pathogens includingVibrio cholerae, have severe limitations when used to estimate viable populations of these organisms in the aquatic environment. By combining the methods of immunofluorescent microscopy, acridine orange direct counting, and direct viable counting, with culture methods such as indirect enumeration by most probable number (MPN) estimation and direct plating, it was shown that bothE. coli andV. cholerae undergo a “nonrecoverable” stage of existence, but remain viable. Following 2-week incubations in saltwater (5–25%o NaCl) microcosms, total counts, measured by direct microscopic examination of fluorescent antibody and acridine orange stained cells, remained unchanged, whereas MPN estimates and plate counts exhibited rapid decline. Results of direct viable counting, a procedure permitting estimate of substrate-responsive viable cells by microscopic examination, revealed that a significant proportion of the nonculturable cells were, indeed, viable. Thus, survival of pathogens in the aquatic environment must be re-assessed. The “die-off” or “decay” concept may not be completely valid. Furthermore, the usefulness of the coliform and fecal coliform indices for evaluating water quality for public health purposes may be seriously compromised, in the light of the finding reported here.


Saltwater Fecal Coliform Acridine Orange Viable Population Much Probable Number 
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. 1.
    American Public Health Association (1980) Standard methods for the examination of water and wastewater, 15th ed. American Public Health Association, Inc., Washington, DCGoogle Scholar
  2. 2.
    Andre DA, Weiser HH, Malaney GW (1967) Survival of bacterial enteric pathogens in farm pond water. J Am Water Works Assoc 59:503–508Google Scholar
  3. 3.
    Apel WA, Dugan PR, Filppi JA, Rheins MS (1976) Detection ofThiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining. Appl Environ Microbiol 32:159–165PubMedGoogle Scholar
  4. 4.
    Baross JA, Hanus FJ, Morita RY (1975) Survival of human enteric and other sewage microorganisms under simulated deep sea conditions. Appl Microbiol 30:309–318PubMedGoogle Scholar
  5. 5.
    Bireley LE, Buck JD (1975) Microbiology of a former dredge spoil disposal area. Mar Pollut Bull 6:107–110CrossRefGoogle Scholar
  6. 6.
    Bordner R, Winter J (eds) (1978) Microbiological methods for monitoring the environment. EPA 600/8-78-017, US Environmental Protection Agency, CincinnatiGoogle Scholar
  7. 7.
    Cherry WB, Goldman M, Carski TR (1960) Fluorescent antibody techniques in the diagnosis of communicable diseases. US Department of Health, Education and Welfare. Public Health Service, Bureau of State Services, Communicable Diseases Center, Atlanta, p 32Google Scholar
  8. 8.
    Coleman WG Jr, Leive L (1979) Two mutations which affect the barrier function of theEscherichia coli K-12 outer membrane. J Bacteriol 139:899–910PubMedGoogle Scholar
  9. 9.
    Colwell RR, Kaper J (1977)Vibrio species as bacterial indicators of potential health hazards associated with water. In: Hoadley AW, Dutka BJ (eds) Bacterial indicators/health hazards associated with water. ASTM STP 635, American Society for Testing and Materials, Philadelphia, pp 115–125Google Scholar
  10. 10.
    Colwell RR, Seidler RJ, Kaper J, Joseph SW, Garges S, Lockman H, Maneval D, Bradford H, Roberts N, Remmers E, Huq I, Huq A (1981) Occurrence ofVibrio cholerae serotype 01 in Maryland and Louisiana estuaries. Appl Environ Microbiol 41:555–558PubMedGoogle Scholar
  11. 11.
    Costerton JW, Ingram JM, Cheng K-J (1974) Structure and function of the cell envelope of gram-negative bacteria. Bacteriol Rev 38:87–110PubMedGoogle Scholar
  12. 12.
    Daley RJ, Hobbie JE (1975) Direct counts of aquatic bacteria by a modified epi-fluorescent technique. Limnol Oceanogr 20:875–882Google Scholar
  13. 13.
    Fliermans CB, Schmidt EL (1975) Autoradiography and immunofluorescence combined for autecological study of single cell activity withNitrobacter as a model system. Appl Microbiol 30:676–684PubMedGoogle Scholar
  14. 14.
    Francisco DE, Mah RA, Rabin AC (1973) Acridine orange epifluorescence technique for counting bacteria in natural waters. Trans Am Microscopical Soc 92:416–421Google Scholar
  15. 15.
    Hobbie JE, Daley RJ, Jasper S (1977) Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol 33:1225–1228PubMedGoogle Scholar
  16. 16.
    Kogure K, Simidu U, Taga N (1979) A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 25:415–420PubMedGoogle Scholar
  17. 17.
    LaLiberte P, Grimes DJ (1982) Survival ofEscherichia coli in lake bottom sediment. Appl Environ Microbiol 43:623–628PubMedGoogle Scholar
  18. 18.
    McFeters GA, Stuart DG (1972) Survival of coliform bacteria in natural waters: Field and laboratory studies with membrane-filter chambers. Appl Microbiol 24:805–811PubMedGoogle Scholar
  19. 19.
    Mitchell R (1968) Factors affecting the decline of non-marine microorganisms in seawater. Water Res 2:534–543CrossRefGoogle Scholar
  20. 20.
    Reed WM, Dugan PR (1978) Distribution ofMethylomonas methanica andMethylosinus trichosporium in Cleveland Harbor as determined by an indirect fluorescent antibody-membrane filter technique. Appl Environ Microbiol 35:422–430Google Scholar
  21. 21.
    Schmidt EL, Bankole RD, Bohilool BB (1968) Fluorescent-antibody approach to study of rhizobia in soil. J Bacteriol 95:1987–1992PubMedGoogle Scholar
  22. 22.
    Singleton FL, Attwell RW, Jangi MS, Colwell RR (1981) Influence of salinity and organic nutrient concentration on survival and growth ofVibrio cholerae in aquatic microcosms. Appl Environ Microbiol 43:1080–1085Google Scholar
  23. 23.
    Strayer RF, Tiedje JM (1978) Application of the fluorescent-antibody technique to the study of a methanogenic bacterium in lake sediments. Appl Environ Microbiol 35:192–198PubMedGoogle Scholar
  24. 24.
    Ward BB, Perry MJ (1980) Immunofluorescent assay of the marine ammonium-oxidizing bacteriumNitrosococcus oceanus. Appl Environ Microbiol 39:913–918Google Scholar

Copyright information

© Springer-Verlag 1982

Authors and Affiliations

  • Huai -Shu Xu
    • 1
  • N. Roberts
    • 2
  • F. L. Singleton
    • 3
  • R. W. Attwell
    • 4
  • D. J. Grimes
    • 5
  • R. R. Colwell
    • 5
  1. 1.Department of Marine BiologyShandong College of OceanographyQingdaoPRC
  2. 2.Lake Charles Regional LaboratoryLake CharlesUSA
  3. 3.Department of Biological SciencesOld Dominion UniversityNorfolkUSA
  4. 4.Department of Biological SciencesManchester PolytechnicManchesterEngland
  5. 5.Department of MicrobiologyUniversity of MarylandCollege ParkUSA

Personalised recommendations