Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate
2012
We describe a simplified microplate
most-probable-number(MPN) procedure to quantify the bacterial
naphthalenedegrader population in soil samples. In this method, the sole substrate
naphthaleneis 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 oiland 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
naphthalenewas dissolved in an adequate concentration to support the growth of
naphthalenedegraders 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
naphthalenedegraders detected in soil.
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