A new direct microscopy based method for evaluating in-situ bioremediation

A new epifluorescent microscopy based method using 5-cyano-2,3-ditolyl tetr azolium chloride (CTC) and 5-(4,6-dichlorotriazinyl) aminofluoroscein (DTAF ) was developed for quantifying total microbial biomass and evaluating leve ls of microbial activity. CTC is a tetrazolium dye that forms fluorescent i ntracellular formazan when biologically reduced by components of the electr on transport system and/or dehydrogenases of metabolically active bacteria. DTAF is a fluorescein-based fluorochrome that selectively stains bacterial cell walls thereby enabling quantification of total bacterial biomass. CTC can be used in conjunction with DTAF to provide the optical resolution nec essary to differentiate metabolically active cells from inactive cells in m icrobial populations associated with subsurface soils. The CTC/DTAF stainin g method has been shown to be effective for quantifying the metabolic activ ity of not only aerobic bacteria, but also diverse groups of anaerobic bact eria. This method allows for the rapid quantification of total and active b acterial numbers in complex soil samples without enrichment or cell elution . In this study, CTC/DTAF staining was applied to evaluate in-situ microbia l activity in petroleum hydrocarbon contaminated subsurface soils from Site s 3 and 13 at Alameda Point, CA. At each site, subsurface microbial activit y at two locations within contaminated plumes were examined and compared to activity at two geologically similar but uncontaminated background locatio ns. Significant bacterial populations were detected in all soils examined, and the biomass estimates were several orders of magnitude higher than thos e obtained by conventional culture-based techniques. Both the total bacteri al concentrations and the numbers of active bacteria in soils from contamin ated areas were substantially higher than those observed in soils from back ground locations. Additionally, the percentages of metabolically active bac teria in the contaminated areas were consistently higher than those detecte d in background areas, suggesting that the enhanced microbial activity was due to microbial contaminant degradation. Although conventional heterotroph ic plate counts failed to show significant microbial activity at either of the sites, soil gas carbon dioxide and methane measurements confirmed that hydrocarbon contaminant degradation was occurring in both areas. The CTC/DT AF staining protocol proved to be a rapid, reliable, and inexpensive method to evaluate the progress of in-situ bioremediation. (C) 1999 Elsevier Scie nce B.V. All rights reserved.