Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.     

Enumeration of water-borne bacteria using viability assays and flow cytometry: a comparison to culture-based techniques

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.     

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 10⁵ bacteria ml⁻¹. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 10³ active AOB ml⁻¹ were detected in the membrane-intact fraction, and 1.40 × 10⁴ active AOB ml⁻¹ were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml⁻¹ detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.     

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.     

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.     

Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and screening of metal-resistant, potentially plant growth-promoting bacteria

This study investigates the impact of long-term heavy metal contamination on the culturable, heterotrophic, functional and genetic diversity of rhizobacterial communities of perennial grasses in water meadow soil. The culturable heterotrophic diversity was investigated by colony appearance on solid LB medium. Genetic diversity was measured as bands in denaturing gradient gel electrophoresis (DGGE) obtained directly from rhizosphere soil and rhizoplane DNA extracts, and from the corresponding culturable communities. In the two rhizospheric fractions the DGGE profiles of the direct DNA extracts were similar and stable among replicates, whereas in the enriched cultures the profiles of the fractions differed, but among the replicates they were similar. One hundred isolates were collected into 33 different operational taxonomic units by use of amplified internal transcribed spacers and into 19 heavy metal-resistant phenotypes. The phylogenetic position of strains belonging to 18 operational taxonomic units, representing more than 80% of the isolates, was determined by 16S rRNA gene sequencing. Several heavy metal-resistant strains were isolated from rhizoplane. Finally, metal-resistant rhizobacteria were tested for plant growth-promoting characteristics; some were found to contain 1-aminocyclopropane-1-carboxylic acid deaminase and/or to produce indole acetic acid and siderophores. Two strains resistant to cadmium and zinc, Pseudomonas tolaasii RP23 and Pseudomonas fluorescens RS9, had all three plant growth-promoting characteristics. Our findings suggest that bacteria can respond to soil metal contamination, and the described methodological approach appears promising for targeting potential plant growth-promoting rhizobacteria.     

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.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column

AIMS: To isolate and identify bacteria from a sand filter column capable of degrading the taste and odour compound, geosmin. In doing so, to investigate if these organisms degrade geosmin either individually or if an alternative mechanism is utilized. METHODS AND RESULTS: Geosmin-degrading bacteria from a biologically active sand filter column were enriched by their growth in a minimal medium supplemented with geosmin as the sole carbon source. By day 51, 21.7 mg l(-1) of geosmin had been degraded as determined by solid-phase microextraction gas chromatography/mass spectrometry, and was accompanied by a 2.12 log(10) increase in active bacterial numbers as measured using the BacLight(TM) bacterial viability kit and flow cytometric enumeration. During the onset of geosmin degradation, the predominance of three bacteria, most similar to previously cultured species of Sphingopyxis alaskensis, Novosphingobium stygiae and Pseudomonas veronii based on 16S rRNA gene sequences was detected by denaturing gradient gel electrophoresis. Subsequent isolation of these organisms revealed that degradation of geosmin, when present as either the sole carbon source (ranging from 40 ng l(-1) to 20 mg l(-1)) or when spiked into sterile reservoir water (37 and 131 ng l(-1)), occurred only when all three isolates were present. None of the isolates was shown to be capable of degrading geosmin either individually or in any combination of two. CONCLUSIONS: This study has reported, for the first time, the cooperative degradation of geosmin by a consortium comprising three gram-negative bacteria isolated from a biologically active sand filter column. SIGNIFICANCE AND IMPACT OF THE STUDY: These results are important for researchers currently employing molecular-based approaches to further understand the biodegradation of geosmin by bacteria, as such studies may be complicated by the discovery of geosmin degradation occurring by a consortium. This study also advances the knowledge surrounding the types of bacteria capable of degrading the taste and odour compound, as investigations to date regarding this are limited.     

16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Spatial and temporal analysis of the microbial community in slow sand filters used for treating horticultural irrigation water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Changes in Microbial Communities,Including both Uncultured and Culturable Bacteria,with Mid-Ocean Ballast-Water Exchange during a Voyage from Japan to Australia

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships'' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.     

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from <Emphasis Type="Italic">Escherichia coli</Emphasis> populations?

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.     

Bacterial Abundance, Activity, and Viability in the Eutrophic River Warnow, Northeast Germany

The River Warnow is the drinking water source for the city of Rostock. Its eutrophic status is accompanied by high amounts of bacteria, which may reach up to 24 x 10(6) cells mL(-1) as recorded during a seasonal study in 2002. Because the river is eutrophic and also heavily loaded with organic matter, this burden is a problem for drinking water purification, as it must be removed completely to not trigger new bacterial growth in the pipeline network. Therefore, restoration measures in the river have to be planned, and bacteria have to be favored as decomposers. That includes the investigation of the physiological state of bacteria in situ. Viable and active cells in the lower reaches of River Warnow were estimated using a broad set of methods. Intact bacteria were investigated by the LIVE/DEAD BacLight bacterial viability kit, containing a mixture of permeant and impermeant nucleic acid stains. Cells with ribosomes were visualized by fluorescence in situ hybridization with the EUB338 oligonucleotide probe. Intact cells and ribosome-containing bacteria represented 24% of total numbers stained by 4'6,-diamidino-2-phenylindole (DAPI) or 66 and 62%, respectively, in relation to all bacteria visualized by the LIVE/DEAD kit. Both fractions were considered as viable, although the fraction of RIB + bacteria is most likely underestimated by the protocol applied. 5-Cyano-2,3-ditolyltetrazolium chloride (CTC) was applied to mark respiring bacteria. The esterase substrate CellTracker Green 5-chloromethylfluorescein diacetate showed cells with intracellular hydrolytic activity. Whereas 1.5% of DAPI-stained bacteria were observed as respiring, 3.8% exhibited intracellular hydrolytic activity on average. If these active fractions were calculated as the percentages of intact cells, much higher fractions of 5.4% were respiring and 16% hydrolytic. Temperature was a main factor influencing total and viable cell numbers simultaneously. The results confirm that there are different states of viable and active cells in natural bacterioplankton communities. However, it remains unclear why fractions of viable and active cells were rather low in this eutrophic river in comparison to similar waters. We recommend to carefully address cells as viable in contrast to nonviable, i.e., dead. As viable cells may be active or inactive with respect to many different activities, e.g., substrate uptake, respiration, hydrolysis, and cell deviation, it is necessary to choose the method to visualize active cells according to the question to be answered.     

不同压力条件下油藏内源细菌群落激活过程中变性梯度凝胶电泳分析?

摘要：【目的】了解常压（1 MPa）和高压（10 MPa）条件下内源细菌激活中的细菌群落和结构的变化。【方法】利用胜利油田沾3×24井产出液水样,在常压和高压下进行富集培养后,定时取样,样品用变性梯度凝胶电泳（denature gradient gel electrophoresis,DGGE）技术分析。【结果】通过对内源微生物激活前后DGGE条带的数量和亮度变化分析表明,油藏环境中的细菌在种类和数量上并不丰富,激活剂的加入改变了菌群原有的贫营养环境,从而使一些因营养缺乏生长受抑制细菌得以大量繁殖,菌群结     

Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water

An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.     

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

Aim: To investigate the application of high‐resolution melt (HRM) analysis for rapid species‐level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. Methods and Results: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR‐DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. Conclusion: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. Significance and Impact of the Study: HRM profiling is suggested as an additional approach for identification of DGGE bands.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.     

Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil

In recent years, culture-independent methods have been used in preference to traditional isolation techniques for microbial community analysis. However, it is questionable whether uncultured organisms from a given sample are important for determining the impact of anthropogenic stress on indigenous communities. To investigate this, soil samples were taken from a site with patchy metal contamination, and the bacterial community structure was assessed with a variety of approaches. There were small differences in microscopic epifluorescence bacterial counts. Denaturing gradient gel electrophoresis (DGGE) profiles of 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar. A clone library generated from the most contaminated sample revealed a diverse bacterial community, which showed similarities to pristine soil communities from other studies. However, the proportion of bacteria from the soil samples that were culturable on standard plate-counting media varied between 0.08 and 2.2%, and these values correlated negatively with metal concentrations. The culturable communities from each sample were compared by 16S-DGGE of plate washes and by fatty acid profiling of individual isolates. Each approach indicated that there were considerable differences between the compositions of the culturable communities from each sample. DGGE bands from both culture-based and culture-independent approaches were sequenced and compared. These data indicated that metal contamination did not have a significant effect on the total genetic diversity present but affected physiological status, so that the number of bacteria capable of responding to laboratory culture and their taxonomic distribution were altered. Thus, it appears that plate counts may be a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.