Note Screening method for detection of hydrocarbon-oxidizing bacteria in oil-contaminated water and soil specimens Petrović Olga a, ⁎, Knežević Petar a , Marković Jelena a , Rončević Srdjan b a Faculty of Sciences, Department of Biology and Ecology, Trg D. Obradovica 5, 21000 Novi Sad, Serbia b Faculty of Sciences, Department of Chemistry, Trg D. Obradovica 3, 21000 Novi Sad, Serbia ABSTRACT ARTICLE INFO Article history: Received 17 January 2008 Received in revised form 31 March 2008 Accepted 31 March 2008 Available online 11 April 2008 Keywords: Activity Bacterial count Hydrocarbon-oxidizing bacteria Oil TTC Toxic action of crude oil on the living world and ecosystems in general is a global problem of both aquatic and terrestrial environments. Bearing in mind the possibility of biodegradation of this toxicant, the procedures of determining counts and activity of cultivable microorganisms, and especially of bacteria responsible for degradation processes, are of great significance. The aim of this work was to study the possibility of modifying some solid media by adding triphenyltetrazolium chloride reagent as an indicator of the dehydrogenase activity, to develop a simple screening method for a simultaneous assessment of the count and activity of cultivable hydrocarbon-oxidizing bacteria in the oil-contaminated environments. The modified method appeared to be rapid and very suitable for the intended purposes. © 2008 Elsevier B.V. All rights reserved. Contamination and degradation of the environment caused by oil hydrocarbons and their derivatives represent a great problem because of the toxic action on the living world and functioning of ecosystems (Jacobuci et al., 2001). However, like many other pollutants, oil undergoes degradation, which may take place by biotic and abiotic processes. For removal of oil contamination recently has been used various remediation techniques that are, to a smaller or greater extent, based on biodegradation. The biodegradation efficiency relies upon microorganisms, first of all on bacteria that are adapted to grow in an oil-contaminated environment, using oil as a source of carbon and energy (Wyszkowska and Kucharski, 2001; Atlas, 1981; Ron and Rosenberg, 2002). Hydrocarbon-oxidizing microorganisms that are capable of degrading hydrocarbons originated from oil are widespread in the ecosystems. However, because of the complex nature of oil and its derivatives as contaminants, the estimation of the count and activity of this group of microorganisms is not always an easy task (Atlas, 1981; Li et al., 2000). Microbiological assessment of the bioremediation potential of water and soil contaminated with oil assumes the application of diverse methods, among which the determination of the counts of those microorganisms that are capable of degrading oil components and the determination of dehydrogenase activity (DHA) of microorganisms have a dominant role. The determination of the count of hydrocarbon-degrading bacteria usually assumes their cultivation on a medium that contains crude oil or refined oil derivatives as a selective medium (Walker et al., 1976). Many researchers dealing with oil-contaminated environments determine the count of hydrocarbon-oxidizing bacteria in liquid media as a most probable number (for instance Gunkel and Trekel, 1967), whereas the others use solid agarised media (e.g. Walker et al., 1976; Adebusoye et al., 2006). The majority of media are of mineral composition, without readily uptakable organic matter (Gunkel and Trekel, 1967; Adebusoye et al., 2006). On the other hand, some media contain small amounts of organic matter such as peptones and yeast extract (Walker et al., 1976). Irrespective of the basic composition of the medium, the main source of carbon is always a mixture of alkanes (oil) (e.g. Delille et al., 2002 use Arabian light crude oil; Walker et al. (1976) use 0.5% oil, etc.), or one particular hydrocarbon (for example, Gunkel and Trekel (1967) use n-hexadecane). The application of oil powder from paraffinic oil base blurs significantly the medium, hindering thus the possibility of discerning small and colorless colonies. Because of that, some authors add 0.003% phenol red to the medium to enhance the contrast between the colonies and medium and thus facilitate counting (Walker et al., 1976). Oxidation of organic matter by microorganisms assumes the involvement of dehydrogenase enzymatic system, so that its activity is a good indicator of the microbiological action in the contaminated environment, as well as of the potential and dynamics of bioremedia- tion. The DHA is measured by colorimetric method using triphenylte- trazolium chloride (TTC) as electron acceptor, the reduction of which Journal of Microbiological Methods 74 (2008) 110–113 ⁎ Corresponding author. E-mail address: petrovico@ib.ns.ac.yu (P. Olga). 0167-7012/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2008.03.012 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth