Indirect Immunofluorescence and FISH for Enumerating Contaminated Site Sulfate-Reducers

A new species of sulfate-reducer, Desulfosporosinus meridiei, was recently isolated from gasoline-contaminated anaerobic groundwater in which degradation of toluene and other hydrocarbons occurred. Ground-water from inside (three sites) and outside (one site) the contaminant plume was probed with specific polyclonal antibodies raised against two strains of D. meridiei (strains T2 and S6). Molecular 16S rRNA probes designed to hybridize with cells of D. meridiei were also used. Cell counts using antistrain T2 antibodies (specific for all strains of D. meridiei and two strains of D. orientis) were similar (10³ cells/mL) both inside and outside the plume as were total DAPI counts (10⁶ cells/mL). The numbers of cells stained with antibodies specific for Group B strains of D. meridiei varied between locations. The molecular probes DSP477A and DSP477B were designed for, and were effective on, pure cultures of D. meridiei and were able to distinguish this species from Desulfovibrio desulfuricans in hybridization experiments. No cells were seen to hybridize with probe DSP477B in groundwater samples. Cell counts in groundwater using the universal eubacterial probe, EUB338, were only 8% to 29% of DAPI counts. Fluorescence intensity was poor and auto fluorescence of particles made counting difficult. This study showed that molecular probing using techniques commonly employed in many laboratories was of little use for evaluating microbial populations in this groundwater. Polyclonal antibodies were considerably more useful for identifying populations of specific cells. The lack of difference in cell numbers between contaminated and nearby uncontami-nated groundwater suggests that cell counts will not always be useful as indicators of intrinsic remediation. sulfate-reducing bacteriaindirect immunofluorescencefluorescent in situ hybridizationbioremediationBTEX contaminationgroundwater.