Skip to main content
SpringerLink
Log in

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

  • Published:
Journal of Industrial Microbiology

Abstract

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alexander M. 1982. Most probable number method for microbial populations. In: Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties, 2nd edn (Page AL, RH Miller and DR Keeney, eds), pp 815–820, American Society for Agronomy and Soil Science Society of America, Madison, WI.

    Google Scholar 

  2. Atlas RM. 1979. Measurement of hydrocarbon biodegradation potentials and enumeration of hydrocarbon-utilizing microorganisms using carbon-14 hydrocarbon-spiked crude oil. In: Native Aquatic Bacteria: Enumeration, Activity, and Ecology, ASTM STP 695 (Costerton JW and RR Colwell, eds), pp 196–204, American Society for Testing and Materials, Philadelphia, PA.

    Google Scholar 

  3. Brown EJ and JF Braddock. 1990. Sheen screen, a miniaturized Mosto-Probable-Number method for the enumeration of oil-degrading microorganisms. Appl Environ Microbiol 56: 3895–3896.

    Google Scholar 

  4. Draper NR and H Smith. 1981. Applied Regression Analysis. John Wiley and Sons, New York.

    Google Scholar 

  5. Gutnick DL and E, Rosenberg. 1977. Oil tankers and pollution: a microbiological approach. Ann Rev Microbiol 31: 379–396.

    Google Scholar 

  6. Herbert RA. 1990. Methods for enumerating microorganisms and determining biomass in natural environments. In: Methods in Microbiology, Vol 22 (Grigorova R and JR Norris, eds), pp 1–39, Academic Press, London.

    Google Scholar 

  7. Jannasch HW and GE Jones. 1959. Bacterial populations in sea water as determined by different methods of enumeration. Limnol Oceanogr 4: 128–139.

    Google Scholar 

  8. Klee AJ. 1993. A computer program for the determination of most probable number and its confidence limits. J Microbiol Meth 18: 91–98.

    Google Scholar 

  9. Mills AL, C Breuil and RR Colwell. 1978. Enumeration of petroleumdegrading marine and estuarine microorganisms by the most probable number method. Can J Microbiol 24: 552–557.

    Google Scholar 

  10. Mulkins-Phillips GJ and JE Stewart. 1974. Distribution of hydrocarbon-utilizing bacteria in Northwestern Atlantic waters and coastal sediments. Can J Microbiol 20: 955–962.

    PubMed  Google Scholar 

  11. Packard TT 1971. The measurement of respiratory electron-transport activity in marine phytoplankton. J Marine Res 29: 235–244.

    Google Scholar 

  12. Rosenberg M and E Rosenberg. 1981 Role of adherence in growth ofAcinetobacter calcoaceticus RAG-1 on hexadecane. J Bacteriol 148: 51–57.

    PubMed  Google Scholar 

  13. Roubal GE and RM Atlas. 1978. Distribution of hydrocarbon-utilizing microorganisms and hydrocarbon biodegradation potentials in Alaskan continental shelf areas. Appl Environ Microbiol 35: 897–905.

    PubMed  Google Scholar 

  14. Roubal GE and RM Atlas. 1979. Hydrocarbon biodegradation in Cook Inlet, Alaska. Dev Ind Microbiol 20: 497–502.

    Google Scholar 

  15. Rowe R, R Todd and J Waide. 1977. Microtechnique for most-probable-number analysis. Appl Environ Microbiol 33: 675–680.

    Google Scholar 

  16. Sexstone AJ and RM Atlas. 1977. Response of microbial populations in Arctic tundra soils to crude oil. Can J Microbiol 23: 1327–1333.

    PubMed  Google Scholar 

  17. Sokal RR and FJ Rohlf. 1981. Biometry, 2nd edn WH Freeman and Company, San Francisco.

    Google Scholar 

  18. Song HG and R Bartha. 1990. Effects of jet fuel spills on the microbial community of soil. Appl Environ Microbiol 56: 646–651.

    Google Scholar 

  19. Thomas HA. 1955. Statistical analysis of coliform data. Sewage Indust Wastes 27: 212–222.

    Google Scholar 

  20. Walker JD and RR Colwell. 1976. Enumeration of petroleum-degrading microorganisms. Appl Environ Microbiol 31: 198–207.

    PubMed  Google Scholar 

Download references

Author information

Author notes

  1. K L Strohmeier

    Present address: Environmental Technologies & Solutions, Inc., 820 Main Street, 41011-1357, Covington, KY, USA

  2. R T Herrington

    Present address: Parsons Engineering Science, Inc, Suite 900, 1700 Broadway, 80290, Denver, CO, USA

Authors and Affiliations

  1. National Risk Management Research Laboratory, US Environmental Protection Agency, 26 W Martin Luther King Dr, 45268, Cincinnati, OH, USA

    J R Haines & A D Venosa

  2. Department of Civil and Environmental Engineering, University of Cincinnati, 26 W Martin Luther King Dr, 45268, Cincinnati, OH, USA

    B A Wrenn, E L Holder, K L Strohmeier & R T Herrington

Authors
  1. J R Haines
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B A Wrenn
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E L Holder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K L Strohmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R T Herrington
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A D Venosa
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haines, J.R., Wrenn, B.A., Holder, E.L. et al. Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure. Journal of Industrial Microbiology 16, 36–41 (1996). https://doi.org/10.1007/BF01569919

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01569919

Keywords

Search

Navigation