Application of the new generation tetrazolium salt (XTT) for the enumeration of hydrocarbon degrading microorganisms using the most probable number method

The objective of this study was to evaluate the performance of two tetrazolium indicators: a traditional one: INT and a new generation one: XTT, for the estimation of hydrocarbon (HC) degrading microorganism's density using the Most Probable Number Technique (MPN). Ninety six composite soil samples were taken and analyzed from Ecorregión Cafetera Colombiana. Degrading microorganisms were recovered in minimum salt medium with saturated HC atmosphere. Degrading HC capacity of the microorganisms was confirmed by successive subcultures in the same medium using diesel as only carbon source. Counts obtained with the two salts were not significantly different (Student t test, p<0,05) but XTT allowed an easier visualization of positive wells due to product solubility of the reduce product. A greater percentage of isolates was obtained using XTT (67%), which suggests that salt type is relevant for recovering of these microorganisms. Additionally, cell detection limit, optimal conditions of XTT concentration and incubation times for detection of activity were evaluated. This evaluation was performed by means of microplate format for hydrocarbon degrading microorganisms using Acinetobacter sp. An inhibitory effect was observed in the recovering of cultivable cells when XTT concentrations increased from 0,5 mM to 2 mM. Incubation time favored this recovering. Detection limit of this technique was established at 10 ² UFC/ml. Production of the XTT-formazan was positively related with initial cell concentration and negatively with incubation time.