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The impact of multi-well plate automation on bacterial flow cytometric analyses was investigated. Cell concentrations in up to 96 samples can be measured accurately, as long as a reproducible staining protocol and a total measurement time of below 80 min is used. Fluorescence distribution in the samples may, however, display some variability.

Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50 W/m 2 . Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7 h −1 , 0.5 h −1 and 0.3 h −1 were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells ( D = 0.05 h −1 ) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3 h −1 only a small fraction (⩽1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70 h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.

The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of −2 was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of ∼1500 kJ m−2, and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m−2. Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m−2 solar UVA (corresponding to 530 W m−2 global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the ‘agony’ of E. coli when it is stressed with sunlight.

Accurate and sensitive online detection tools would benefit both fundamental research and practical applications in aquatic microbiology. Here, we describe the development and testing of an online flow cytometer (FCM), with a specific use foreseen in the field of drinking water microbiology. The system incorporated fully automated sampling and fluorescent labeling of bacterial nucleic acids with analysis at 5-min intervals for periods in excess of 24 h. The laboratory scale testing showed sensitive detection (< 5% error) of bacteria over a broad concentration range (1 × 10(3) -1 × 10(6) cells mL(-1) ) and particularly the ability to track both gradual changes and dramatic events in water samples. The system was tested with bacterial pure cultures as well as indigenous microbial communities from natural water samples. Moreover, we demonstrated the possibility of using either a single fluorescent dye (e.g., SYBR Green I) or a combination of two dyes (SYBR Green I and Propidium Iodide), thus broadening the application possibilities of the system. The online FCM approach described herein has considerable potential for routine and continuous monitoring of drinking water, optimization of specific drinking water processes such as biofiltration or disinfection, as well as aquatic microbiology research in general.

Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation: apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m−2) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (−2).

Fast and accurate monitoring of chemical and microbiological parameters in drinking water is essential to safeguard the consumer and to improve the understanding of treatment and distribution systems. However, most water utilities and drinking water guidelines still rely solely on time-requiring heterotrophic plate counts (HPC) and plating for faecal indicator bacteria as regular microbiological control parameters. The recent development of relative simple bench-top flow cytometers has made rapid and quantitative analysis of cultivation-independent microbial parameters more feasible than ever before. Here we present a study using a combination of cultivation-independent methods including fluorescence staining (for membrane integrity, membrane potential and esterase activity) combined with flow cytometry and total adenosine tri-phosphate (ATP) measurements, to assess microbial viability in drinking water. We have applied the methods to different drinking water samples including non-chlorinated household tap water, untreated natural spring water, and commercially available bottled water. We conclude that the esterase-positive cell fraction, the total ATP values and the high nucleic acid (HNA) bacterial fraction (from SYBR Green I staining) were most representative of the active/viable population in all of the water samples. These rapid methods present an alternative way to assess the general microbial quality of drinking water as well as specific events that can occur during treatment and distribution, with equal application possibilities in research and routine analysis.

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli , Salmonella enterica serovar Typhimurium, Shigella flexneri , and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis , which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.