A tentative direct microscopic method for counting living marine bacteria.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.     

The application of viable count procedures for measuring viable cells in the various marine environments

Aims: To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony‐forming units and the contribution of capsule‐bearing bacteria to the total number of bacteria and esterase‐active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Methods and Results: Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0–30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l^?1 (at 35°C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l^?1 for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. Conclusions: The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. Significance and Impact of the Study: The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.     

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization,direct viable counting and solid phase cytometry

Aims: We developed an improved Fluorescent In Situ Hybridization FISH‐based method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. Methods and Results: The method includes a direct viable count (DVC) assay, multi‐probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC–FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. Conclusion: The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. Significance and Impact of the Study: The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments.     

Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples.

Sewage effluent and outfall confluence samples were collected at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico; outfall confluence samples at Ocean City, Md., were also collected. Samples from uncontaminated open ocean areas served as clean-water controls. Bacteria were enriched in marine broth 2216 amended with 1 microgram of one of a set of chemicals selected for study per ml: nitrobenzene, dibutyl phthalate, m-cresol, o-cresol, 4-nitroaniline, bis(tributyltin) oxide, and quinone. MICs of the chemicals were determined individually for all isolates. Bacterial isolates were evaluated for resistance to nine different antibiotics and for the presence of plasmid DNA. Treated sewage was found to contain large numbers of bacteria simultaneously possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin, erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples, but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic sewage-impacted waters or from uncontaminated open ocean sites.     

Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples

Sewage effluent and outfall confluence samples were collected at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico; outfall confluence samples at Ocean City, Md., were also collected. Samples from uncontaminated open ocean areas served as clean-water controls. Bacteria were enriched in marine broth 2216 amended with 1 microgram of one of a set of chemicals selected for study per ml: nitrobenzene, dibutyl phthalate, m-cresol, o-cresol, 4-nitroaniline, bis(tributyltin) oxide, and quinone. MICs of the chemicals were determined individually for all isolates. Bacterial isolates were evaluated for resistance to nine different antibiotics and for the presence of plasmid DNA. Treated sewage was found to contain large numbers of bacteria simultaneously possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin, erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples, but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic sewage-impacted waters or from uncontaminated open ocean sites.     

Formation of nonculturable Escherichia coli in drinking water

AIMS: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. METHODS AND RESULTS: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. CONCLUSIONS: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity on loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity no loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.     

Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.     

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.     

Short-term variations of bacterioplankton in Antarctic zone: Terre Adelie area

Previous studies on Antarctic seawater have demonstrated the presence of significant numbers of bacteria, but their in situ activity has not been demonstrated. In order to demonstrate this hypothetical activity, a scheduled survey was conducted from January to February 1986 in a coastal area of Adelie Land. Seawater samples were collected in a selected station every day or every hour during a 17 hour period. Bacterial communities in each sample were studied by measuring direct and viable counts, frequency of dividing cells estimation, taxonomic analysis, and heterotrophic potential. Complementary studies used batch cultures with artificial nutrient supplements. The results clearly suggest a strong potential activity of the natural Antarctic bacterial microflora.     

Microbiological Comparison of Surface Soil and Unsaturated Subsurface Soil from a Semiarid High Desert

Thirty-two chemoheterotrophic bacteria were isolated from unsaturated subsurface soil samples obtained from ca. 70 m below land surface in a high desert in southeastern Idaho. Most isolates were gram positive (84%) and strict aerobes (79%). Acridine orange direct counts of microbes in one subsurface sample showed lower numbers than similar counts performed on surface soils from the same location (ca. 5 × 10⁵ versus 2 × 10⁶ cells per g [dry weight] of soil), but higher numbers than those from plate counts performed on the subsurface material. Another sample taken from the same depth at another location showed no evidence of colonies under identical conditions. Soil analyses indicated that subsurface sediments versus surface soils were slightly alkaline (pH 7.9 versus 7.4), had a higher water content (25.7 versus 6.3%), and had lower organic carbon concentrations (0.05 to 0.17 versus 0.25% of soil dry weight). Analyses of biologically relevant gases from the unsaturated subsurface indicated an aerobic environment. As in other unsaturated soil environments, either a high proportion of bacteria in these subsurface sediments are not viable or they are incapable of growth on conventional media under aerobic conditions. The presence and numbers of bacteria in these deep sediments may be influenced by colonization opportunities afforded by periodic percolation of surface water through fractures in overlying strata.     

Effect of Kepone on estuarine microbial activity

In situ heterotrophic uptake of mixed¹⁴C-amino acids and direct viable cell (DVC) count of Chesapeake Bay water samples were not significantly affected by the insecticide Kepone at concentrations 0.01 mg/1. Maximum inhibition of heterotrophic uptake,ca. 85–90%, and DVC count, 45–97%, was evident at concentrations of Kepone exceeding 0.2 mg/1. A specific activity index (Metabolic Activity/DVC or Kepone-resistant DVC), heterotrophic uptake, and DVC count were found to be statistically correlated (a=0.05) to one another, but negatively correlated with concentration of Kepone. The direct viable cell count proved to be a rapid, simple method for estimating the effect of Kepone on in situ estuarine microbial activity.     

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.     

Characterization and resuscitation of viable but nonculturable <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> VIB283

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4°C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10¹⁰ to 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.     

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.     

Comparison of bacteria from deep subsurface sediment and adjacent groundwater

Samples of groundwater and the enclosing sediments were compared for densities of bacteria using direct (acridine orange direct staining) and viable (growth on 1% PTYG medium) count methodology. Sediments to a depth of 550 m were collected from boreholes at three sites on the Savannah River Site near Aiken, South Carolina, using techniques to insure a minimum of surface contamination. Clusters of wells screened at discreet intervals were established at each site. Bacterial densities in sediment were higher, by both direct and viable count, than in groundwater samples. Differences between direct and viable counts were much greater for groundwater samples than for sediment samples. Densities of bacteria in sediment ranged from less than 1.00×10⁶ bacteria/g dry weight (gdw) up to 5.01 ×10⁸ bacteria/gdw for direct counts, while viable counts were less than 1.00×10³ CFU/gdw to 4.07×10⁷ CFU/gdw. Bacteria densities in groundwater were 1.00×10³–6.31×10⁴ bacteria/ml and 5.75–4.57×10² CFU/ml for direct and viable counts, respectively. Isolates from sediment were also found to assimilate a wider variety of carbon compounds than groundwater bacteria. The data suggest that oligotrophic aquifer sediments have unique and dense bacterial communities that are attached and not reflected in groundwater found in the strata. Effective in situ bioremediation of contaimination in these aquifers may require sampling and characterization of sediment communities.