Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel

Site selection for a spent nuclear fuel (SNF) repository required analysis of microbial abundance and diversity at two Swedish sites, Forsmark and Laxemar-Simpevarp. Information about sulphate-reducing bacteria (SRB) was required, as sulphide could corrode copper SNF canisters. Total number of cells (TNC) and ATP were analysed, and plate counts and most probable number (MPN) analyses were conducted using eight media based on different electron donors and acceptors for specific microorganism physiological groups. Groundwater chemical composition and E(h) were analysed; sampling depths were 112-978 m below sea level. TNC was 5.5 × 10(3) to 4.7 × 10(5) cells mL(-1), correlating with ATP concentrations. Culturability in TNC percentage was 0.01-35.9, averaging 5.12. Culturable numbers varied greatly between sample positions and uncorrelated with depth. SRB were found in 29 samples and were below detection in three; the MPN of SRB correlated negatively with E(h), as did the MPN of acetogens. Data indicated that microbial sulphate reduction was ongoing in many sampled aquifers; published stable isotope data and modelling results supported this observation. The sites did not differ significantly, but the large data range suggested that analysis of more samples would enable detailed evaluation of microbial processes and their relationship with geochemical information.     

Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10¹ and 10⁵ cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Applying a most probable number method for enumerating planktonic, dissimilatory, ammonium-producing, nitrate-reducing bacteria in oil field waters

A most probable number (MPN) method was used to enumerate dissimilatory ammonium-producing, nitrate-reducing bacteria (DAP-NRB) in oil field waters and to determine whether they were stimulated by nitrate addition used to control hydrogen sulfide production. An ammonium production medium with 5 carbon and energy sources (acetate, glucose, glycerol, pyruvate, and succinate) and nitrate was used in a 3-tube MPN procedure to enumerate DAP-NRB. These bacteria were detected in 12 of 18 oil field water samples, but they were seldom detected in wellhead samples. Three oil field water samples were amended with nitrate in serum bottles and the numbers of different NRB were determined over a 38-day incubation time. This amendment stimulated increases in the numbers of heterotrophic NRB and autotrophic nitrate-reducing, sulfide-oxidizing bacteria, but DAP-NRB remained a minor portion of these communities. Overall, DAP-NRB were present in many of the oil field waters that were examined but their numbers were low. It appears that DAP-NRB would play a minor role in the consumption of nitrate injected into oil field waters for the control of hydrogen sulfide production.     

Failure of the most-probable-number technique to detect coliforms in drinking water and raw water supplies.

A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Enumeration, isolation, and characterization of n(2)-fixing bacteria from seawater

Marine pelagic N(2)-fixing bacteria have not, in general, been identified or quantified, since low or negligible rates of N(2) fixation have been recorded for seawater when blue-green algae (cyanobacteria) are absent. In the study reported here, marine N(2)-fixing bacteria were found in all samples of seawater collected and were analyzed by using a most-probable-number (MPN) method. Two different media were used which allowed growth of microaerophiles, as well as that of aerobes and facultative anaerobes. MPN values obtained for N(2)-fixing bacteria ranged from 0.4 to 1 x 10 per liter for water collected off the coast of Puerto Rico and from 2 to 5.5 x 10 per liter for Chesapeake Bay water. Over 100 strains of N(2)-fixing bacteria were isolated from the MPN tubes and classified, yielding four major groups of NaCl-requiring bacteria based on biochemical characteristics. Results of differential filtration studies indicate that N(2)-fixing bacteria may be associated with phytoplankton. In addition, when N(2)-fixing bacteria were inoculated into unfiltered seawater and incubated in situ, nitrogenase activity could be detected within 1 h. However, no nitrogenase activity was detected in uninoculated seawater or when bacteria were incubated in 0.2-mum-filtered (phytoplankton-free) seawater. The ability of these isolates to fix N(2) at ambient conditions in seawater and the large variety of N(2)-fixing bacteria isolated and identified lead to the conclusion that N(2) fixation in the ocean may occur to a greater degree than previously believed.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Diversity of Ferrous Iron-Oxidizing,Nitrate-Reducing Bacteria and their Involvement in Oxygen-Independent Iron Cycling

In previous studies, three different strains (BrG1, BrG2, and BrG3) of ferrous iron-oxidizing, nitrate-reducing bacteria were obtained from freshwater sediments. All three strains were facultative anaerobes and utilized a variety of organic substrates and molecular hydrogen with nitrate as electron acceptor. In this study, analyses of 16S rDNA sequences showed that strain BrG1 was affiliated with the genus Acidovorax, strain BrG2 with the genus Aquabacterium, and strain BrG3 with the genus Thermomonas. Previously, bacteria similar to these three strains were detected with molecular techniques in MPN dilution series for ferrous iron-oxidizing, nitrate-reducing bacteria inoculated with different freshwater sediment samples. In the present study, further molecular analyses of these MPN cultures indicated that the ability to oxidize ferrous iron with nitrate is widespread amongst the Proteobacteria and may also be found among the Gram-positive bacteria with high GC content of DNA. Nitrate-reducing bacteria oxidized ferrous iron to poorly crystallized ferrihydrite that was suitable as an electron acceptor for ferric iron-reducing bacteria. Biologically produced ferrihydrite and synthetically produced ferrihydrite were both well suited as electron acceptors in MPN dilution cultures. Repeated anaerobic cycling of iron was shown in a coculture of ferrous iron-oxidizing bacteria and the ferric iron-reducing bacterium Geobacter bremensis. The results indicate that iron can be cycled between its oxidation states +II and +III by microbial activities in anoxic sediments.     

Enumeration and Detection of Anaerobic Ferrous Iron-Oxidizing, Nitrate-Reducing Bacteria from Diverse European Sediments

Anaerobic, nitrate-dependent microbial oxidation of ferrous iron was recently recognized as a new type of metabolism. In order to study the occurrence of three novel groups of ferrous iron-oxidizing, nitrate-reducing bacteria (represented by strains BrG1, BrG2, and BrG3), 16S rRNA-targeted oligonucleotide probes were developed. In pure-culture experiments, these probes were shown to be suitable for fluorescent in situ hybridization, as well as for hybridization analysis of denaturing gradient gel electrophoresis (DGGE) patterns. However, neither enumeration by in situ hybridization nor detection by the DGGE-hybridization approach was feasible with sediment samples. Therefore, the DGGE-hybridization approach was combined with microbiological methods. Freshwater sediment samples from different European locations were used for enrichment cultures and most-probable-number (MPN) determinations. Bacteria with the ability to oxidize ferrous iron under nitrate-reducing conditions were detected in all of the sediment samples investigated. At least one of the previously described types of bacteria was detected in each enrichment culture. MPN studies showed that sediments contained from 1 × 10⁵ to 5 × 10⁸ ferrous iron-oxidizing, nitrate-reducing bacteria per g (dry weight) of sediment, which accounted for at most 0.8% of the nitrate-reducing bacteria growing with acetate. Type BrG1, BrG2, and BrG3 bacteria accounted for an even smaller fraction (0.2% or less) of the ferrous iron-oxidizing, nitrate-reducing community. The DGGE patterns of MPN cultures suggested that more organisms than those isolated thus far are able to oxidize ferrous iron with nitrate. A comparison showed that among the anoxygenic phototrophic bacteria, organisms that have the ability to oxidize ferrous iron also account for only a minor fraction of the population.     

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO(3)(-) depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl(-) concentrations, matching those in the water samples. Many methods for measuring NO(3)(-) concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl(-) and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO(3)(-) depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl(-) or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.     

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 10⁶ or 10⁹ total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/10⁶-AODC) than in water samples (0.02 to 0.4 PRM/10⁶-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/10⁹-AODC) than from other sites (nondetectable to 0.77 PRM/10⁹-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.     

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.     

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.     

A lectin microarray approach for the rapid analysis of bacterial glycans

Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Bacteria can rapidly alter their glycan coats by switching the genes that are involved on and off in a phase-variable manner. Currently, there is a lack of appropriate tools for studying dynamic carbohydrate alterations. Here, we present a lectin microarray protocol for the high-throughput evaluation of cell-surface microbial sugars. The binding patterns of fluorescent bacteria to these arrays provide a simple means to fingerprint bacteria based on their surface carbohydrates. In addition, this method provides a rapid, parallel evaluation of glycans from multiple bacterial samples, allowing dynamic changes in carbohydrate structures to be studied. The entire procedure takes approximately 12 h but the printing of the microarray can be performed in advance.     

A Method of Profiling Microbial Communities Based on a Most-Probable-Number Assay That Uses BIOLOG Plates and Multiple Sole Carbon Sources

A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.     

A method of profiling microbial communities based on a most-probable-number assay that uses BIOLOG plates and multiple sole carbon sources.

A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.     

Storage of oil field-produced waters alters their chemical and microbiological characteristics

Many oil fields are in remote locations, and the time required for shipment of produced water samples for microbiological examination may be lengthy. No studies have reported on how storage of oil field waters can change their characteristics. Produced water samples from three Alberta oil fields were collected in sterile, industry-approved 4-l epoxy-lined steel cans, sealed with minimal headspace and stored under anoxic conditions for 14 days at either 4°C or room temperature (ca. 21°C). Storage resulted in significant changes in water chemistry, microbial number estimates and/or community response to amendment with nitrate. During room-temperature storage, activity and growth of sulfate-reducing bacteria (and, to a lesser extent, fermenters and methanogens) in the samples led to significant changes in sulfide, acetate and propionate concentrations as well as a significant increase in most probable number estimates, particularly of sulfate-reducing bacteria. Sulfide production during room-temperature storage was likely to be responsible for the altered response to nitrate amendment observed in microcosms containing sulfidogenic samples. Refrigerated storage suppressed sulfate reduction and growth of sulfate-reducing bacteria. However, declines in sulfide concentrations were observed in two of the three samples stored at 4°C, suggesting abiotic losses of sulfide. In one of the samples stored at room temperature, nitrate amendment led to ammonification. These results demonstrate that storage of oil field water samples for 14 days, such as might occur because of lengthy transport times or delays before analysis in the laboratory, can affect microbial numbers and activity as well as water sample chemistry.