, Volume 23, Issue 1, pp 47–55 | Cite as

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

  • Kaisa Wallenius
  • Kaisa Lappi
  • Anu Mikkonen
  • Annika Wickström
  • Anu Vaalama
  • Taru Lehtinen
  • Leena Suominen
Original Paper


We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.


Hydrocarbon degraders MPN Naphthalene Optical density Phase partitioning Soil 



This work was funded by the Academy of Finland, University of Helsinki and Ekokem Oy. We thank Elina Kondo, University of Helsinki, for supervision in the oil analytics and Rannveig Guicharnaud, Agricultural University of Iceland, for supervision in the microbial biomass measurements.

Supplementary material

10532_2011_9485_MOESM1_ESM.xls (26 kb)
Online Resource 1. Naphthalene concentration in uninoculated buffer as a function of distance from the spot source of naphthalene crystals (10 days exposure; 6 replicates). The measurement was performed by UV spectroscopy (280 nm) using a standard curve prepared from serial dilutions of saturated aqueous naphthalene solution. (XLS 26 kb)
10532_2011_9485_MOESM2_ESM.tif (5.9 mb)
Online Resource 2. Representative examples of the substrate plates (A: silicone oil method + INT, B: vapour method) and the negative control plates (C: silicone oil method + INT, D: vapour method) at the time when the results were scored. (TIFF 6025 kb)


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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Kaisa Wallenius
    • 1
  • Kaisa Lappi
    • 1
  • Anu Mikkonen
    • 1
  • Annika Wickström
    • 1
  • Anu Vaalama
    • 1
  • Taru Lehtinen
    • 2
  • Leena Suominen
    • 1
  1. 1.Department of Food and Environmental SciencesUniversity of HelsinkiHelsinkiFinland
  2. 2.Department of Environmental SciencesAgricultural University of IcelandReykjavikIceland

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