Ž . Journal of Hazardous Materials B67 1999 299–312 A new direct microscopy based method for evaluating in-situ bioremediation Vishvesh K. Bhupathiraju a , Mark Hernandez b , Paula Krauter c , Lisa Alvarez-Cohen a, ) a Department of CiÕil and EnÕironmental Engineering, UniÕersity of California, Berkeley, CA 94720-1710, USA b Department of CiÕil, EnÕironmental and Architectural Engineering, UniÕersity of Colorado, Boulder, CO 80309, USA c EnÕironmental Protection Department, Lawrence LiÕermore National Laboratory, L-619, LiÕermore, CA 94550, USA Received 1 November 1998; received in revised form 19 March 1999; accepted 26 March 1999 Abstract A new epifluorescent microscopy based method using 5-cyano-2,3-ditolyl tetrazolium chloride Ž . Ž . Ž . CTC and 5- 4,6-dichlorotriazinyl aminofluoroscein DTAF was developed for quantifying total microbial biomass and evaluating levels of microbial activity. CTC is a tetrazolium dye that forms fluorescent intracellular formazan when biologically reduced by components of the electron transport system andror 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 necessary to differentiate metabolically active cells from inactive cells in microbial populations associated with subsurface soils. The CTCrDTAF staining method has been shown to be effective for quantifying the metabolic activity of not only aerobic bacteria, but also diverse groups of anaerobic bacteria. This method allows for the rapid quantification of total and active bacterial numbers in complex soil samples without enrichment or cell elution. In this study, CTCrDTAF staining was applied to evaluate in-situ microbial activity in petroleum hydrocarbon contaminated subsurface soils from Sites 3 and 13 at Alameda Point, CA. At each site, subsurface microbial activity at two locations within contaminated plumes were examined and compared to activity at two geologically similar but uncontaminated background locations. Significant bacterial populations were detected in all soils examined, and the biomass estimates were several orders of magnitude higher than those obtained by conventional culture-based techniques. Both the total ) Corresponding author. Tel.: q1-510-643-5969; fax: q1-510-642-7483; e-mail: alvarez@ce.berkeley.edu 0304-3894r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0304-3894 99 00045-X