Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Determination of Complement-Mediated Killing of Bacteria by Viability Staining and Bioluminescence

Complement-mediated killing of bacteria was monitored by flow cytometric, luminometric, and conventional plate counting methods. A flow cytometric determination of bacterial viability was carried out by using dual staining with a LIVE/DEAD BacLight bacterial viability kit. In addition to the viable cell population, several other populations emerged in the fluorescence histogram, and there was a dramatic decrease in the total cell count in the light-scattering histogram in the course of the complement reaction. To permit luminometric measurements, Bacillus subtilis and Escherichia coli were made bioluminescent by expressing an insect luciferase gene. Addition of substrate after the complement reaction resulted in bioluminescence, the level of which was a measure of the viable cell population. All three methods gave essentially the same killing rate, suggesting that the bacteriolytic activity of serum complement can be measured rapidly and conveniently by using viability stains or bioluminescence. In principle, any bacterial strain can be used for viability staining and flow cytometric analysis. For the bioluminescence measurements genetically engineered bacteria are needed, but the advantage is that it is possible to screen automatically a large number of samples.     

饮用水微生物的安全快速检测

【目的】为了更好地分析饮用水中的微生物含量。【方法】利用流式细胞术(Flowcytometry,FCM)、ATP测定方法检测瓶装无气饮用水中的微生物数量、可同化有机碳(Assimilable organic carbon,AOC)含量以及微生物活性,并将检测结果与传统的饮用水微生物检测技术相对照。【结果】FCM方法可快速区分水样中的活性细菌和非活性细菌,AOC含量反映了水样中微生物再生能力;而ATP检测方法也能比异养细菌平板计数法(Heterotrophic plate count,HPC)更好地反映瓶装无气饮用水中的实际微生物含量。【结论】FCM、ATP测定方法要明显优于依赖于培养的传统方法。     

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

AIMS: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1) were examined by both flow cytometry and traditional microbiological analyses. The correlation coefficient (r) between the two methods was 0.71. For the differential analyses of the dominant bacterial populations four different parameters were used to give a species-characteristic pattern. The four parameters were as follows: staining with Oregon Green conjugated wheat germ agglutinin that binds to the cell wall of bacteria, staining with hexidium iodide that binds to all bacterial DNA, the flow cytometric forward scatter and the flow cytometric side scatter. Three regions in the flow cytometric plot were defined: region 1 includes bacteria mainly associated with poor hygiene, region 2 includes psychrotrophic hygiene bacteria and region 3 includes bacteria mainly related to mastitis. The ability of the flow cytometric technique to predict the main cause of elevated bacterial counts on routine samples was examined. Comparing these results with results obtained by traditional microbiological analyses for identification showed that for 81% of the samples the two techniques agreed on the main cause of an elevated bacterial count. CONCLUSIONS: The ability of the presented flow cytometric technique to enumerate and differentiate bacteria in bulk tank milk according to the main cause of elevated counts was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study described the first step in development of a technique suitable for routine analyses of bulk tank milk samples. A technique indicating the main cause of an elevated count will enable the farmer to eliminate the contamination source within a short time limit.     

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.     

Direct Measurements of Natural Planktonic Bacterial Community Viability by Flow Cytometry

A range of fluorescent viability dyes were used in conjunction with flow cytometry to rapidly enumerate viable bacteria from freshwater environments. Optimal labelling was achieved by using carboxyfluorescein diacetate or chemchrome B with a detergent-mediated permeabilization step. The viable bacterial count under optimal conditions was 7% in oligotrophic lake water and 75% in polluted river water.     

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ~10⁸ bacteria/ml (equivalent to ~10⁷ CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Profiling bacterial survival through a water treatment process and subsequent distribution system

AIMS: To profile fractions of active bacteria and of bacteria culturable with routine heterotrophic plate count (HPC) methods through a typical water treatment process and subsequent distribution system. In doing so, investigate how water treatment affects both bacterial abundance and diversity, and reveal the identities of active bacteria not detected by traditional HPC culture. METHODS AND RESULTS: Profiling active fractions was performed by flow cytometric cell sorting of either membrane-intact (BacLight kit) or enzymatically active (carboxyfluorescein diacetate, CFDA) bacteria, followed by eubacterial 16S rDNA-directed PCR and denaturing gradient gel electrophoresis (DGGE). Water treatment significantly reduced active bacterial numbers detected by the BacLight kit and CFDA assay by 2.89 and 2.81 log respectively. Bacterial diversity was also reduced from > 20 DGGE bands in the active fractions of reservoir water to only two bands in the active fractions of finished water. These two bands represented Stenotrophomonas maltophila, initially culturable by HPC, and a Burkholderia-related species. Both species maintained measurable traits of physiological activity in distribution system bulk water but were undetected by HPC. CONCLUSIONS: Flow cytometric cell sorting with PCR-DGGE, to assess water treatment efficacy, identified active bacteria from a variety of major phylogenetic groups undetected by routine HPC. Following treatment S. maltophila and a Burkholderia-related species retained activity and entered distribution undetected by HPC. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods used here demonstrate how water treatment operators can better monitor water treatment plant efficacy and assess distribution system instability by the detection and identification of active bacteria recalcitrant to routine HPC culture.     

Comparison of methods for detection and enumeration of airborne microorganisms collected by liquid impingement.

Bacterial agents and cell components can be spread as bioaerosols, producing infections and asthmatic problems. This study compares four methods for the detection and enumeration of aerosolized bacteria collected in an AGI-30 impinger. Changes in the total and viable concentrations of Pseudomonas fluorescens in the collection fluid with respect to time of impingement were determined. Two direct microscopic methods (acridine orange and BacLight) and aerodynamic aerosol-size spectrometry (Aerosizer) were employed to measure the total bacterial cell concentrations in the impinger collection fluid and the air, respectively. These data were compared with plate counts on selective (MacConkey agar) and nonselective (Trypticase soy agar) media, and the percentages of culturable cells in the collection fluid and the bacterial injury response to the impingement process were determined'. The bacterial collection rate was found to be relatively unchanged during 60 min of impingement. The aerosol measurements indicated an increased amount of cell fragments upstream of the impinger due to continuous bacterial nebulization. Some of the bacterial clusters, present in the air upstream of the impinger, deagglomerated during impingement, thus increasing the total bacterial count by both direct microscopic methods. The BacLight staining technique was also used to determine the changes in viable bacterial concentration during the impingement process. The percentage of viable bacteria, determined as a ratio of BacLight live to total counts was only 20% after 60 min of sampling. High counts on Trypticase soy agar indicated that most of the injured cells could recover. On the other hand, the counts from the MacConkey agar were very low, indicating that most of the cells were structurally damaged in the impinger. The comparison of data on the percentage of injured bacteria obtained by the traditional plate count with the data on percentage of nonviable bacteria obtained by the BacLight method showed good agreement.     

Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry

Aims: This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro‐organisms (Escherichia coli and Klebsiella pneumonia). Methods and Results: Two analytical methods were employed; viable plate counts (CFU ml^?1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions: Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and Impact of the study: Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.     

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders

AIMS: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. METHODS AND RESULTS: The BactiFlow flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. CONCLUSIONS: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.     

Online monitoring of Escherichia coli ghost production

Controlled expression of cloned phi X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.     

Evaluation of a commercial fluorochromic system for the rapid detection and estimation of wine lactic acid bacteria by DEFT

A commercial fluorochromic system was evaluated for the rapid detection of lactic acid bacteria in fortified wines by the epifluorescent filter technique (DEFT). The viability test used, employing the fluorescence dyes SYTO 9 and propidium iodide, was able to detect and clearly differentiate viable from non-viable cells (killed with a 50% v/v ethanol solution). A good overall agreement ( r ₌ 0·92) was obtained between the DEFT count and the plate count in the range studied (5 × 10²–4 × 10⁹ cells ml⁻¹). Wine components which might otherwise interfere with the method could be removed by including simple wash steps in the protocol. This measure proved critical to the success of the procedure. For practical purposes, the rapid method studied seems to be a good alternative to the traditional cultural methods as part of quality control programmes in wine making. It may also be useful when studying the efficacy of certain treatments in the elimination of wine bacterial contaminants.     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton

The cell-sorting capability of flow cytometers makes it possible to isolate specific populations of cells with pre-defined cytometric characteristics. A better knowledge of the biological effects of the sorting process is necessary for the future cell sorting applications. In this paper we report the effects of flow cytometric sorting on bacterial viability and exoproteolytic activity (EPA) of bacterial cultures and marine bacterioplankton. Sorting bacterial cultures and bacterioplankton samples reduce viability as assessed by plate counts and produce variations in the exoproteolytic activity. These effects indicate that deflected electrostatic sorting may significantly alter the biological properties of the sorted bacteria.     

Survival of Salmonella Species in River Water

The survival of four Salmonella strains in river water microcosms was monitored by culturing techniques, direct counts, whole-cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytometry. Plate counts of bacteria resuspended in filtered and untreated river water decreased several orders of magnitude within the first week of incubation, while they did not decrease as rapidly in autoclaved water. In situ hybridization studies suggested a rapid decrease in ribosomal content, as determined by the drastic decrease in the number of detectable cells after 72 h. In contrast, direct counts remained relatively constant during 45 days in all microcosoms. Although the culturable counts of two bacterial strains in filtered water after 31 days represented approximately 0.001% of the total counts, direct viable counts and resuscitation studies with a dilution series suggested that the number of viable bacteria was at least four orders of magnitude higher. Additionally, notable changes in forward scatter and in nucleic acid content were observed only after 4 h of nutrient amendments by flow cytometry. However, cells from the resuscitation experiments did not grow on solid media unless cell-free supernatant from viable cultures was added during the resuscitation period. The results in this study suggest the presence of a not immediately culturable status in Salmonella. Received: 20 October 1999 / Accepted: 10 January 2000     

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.     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Application of laser scanning for the rapid and automated detection of bacteria in water samples

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.