Viability of gut microbes as a complementary earthworm biomarker of metal exposure

Earthworms are standard species used in soil ecotoxicology to evaluate the adverse effects of soil contaminants. This study proposes the assessment of the viability of earthworm gut microbes as an indicator in a site-specific test of soil toxicity. Using slow centrifugation, the microbial community was extracted from the guts of earthworms that had been exposed to copper (Cu)- or nickel (Ni)-contaminated soil. Microbial cell viability was assessed using calcein acetoxymethyl ester staining and flow cytometric analysis. We confirmed a metal concentration-dependent decrease in the cell viability of the gut microbial community. The general endpoints, including survival, abnormalities, coelomocyte activity, and metal bioaccumulation, showed a metal concentration-dependent response, and were strongly associated with gut microbial viability in the Ni-exposure group. In contrast, the general endpoints in the Cu-exposure group were significantly different from those in the former group, because the soil penetration rate of the earthworms was very low on the Cu-contaminated soil. Our results indicated that the gut microbial community viability assay holds potential for assessing the toxicity of soil to field worms by simply and rapidly monitoring the viability of the earthworm gut microbial community.     

An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations.

BACKGROUND: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but nonculturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. METHODS: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. RESULTS: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. CONCLUSIONS: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganisms in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article.     

Effects and applications of sub-lethal ultrasound, electroporation and UV radiations in bioprocessing

Advances in bioprocess technology involving microbial cells have led to increased and improved production of beneficial new products and bioactive compounds. However, the semipermeable barrier of the cell membrane often retards the efficient productivity or reaction rate of the cells. Physical treatments such as ultrasound, electroporation and UV radiation provide an efficient approach to increase membrane permeability, leading to enhanced productivity of microbial cells. It is important to note that extensive membrane permeabilization by these physical treatments could be detrimental to cell viability leading to lower yield. An appropriate selection of sublethal dosage and intensity of these physical treatments are critical to preserve the viability of cells and at the same time maintain their bioprocess applications. Despite the promising applications of these physical treatments, safety issues related to possible genotoxicity or mutation of cells upon treatments have been raised. This genotoxic effect of physical treatments could be prevented if appropriate measures are taken, without compromising their bioprocess potentials. The current review highlights the effect of sublethal physical treatments such as ultrasound, electroporation and UV radiation on the viability of cells, their potential bioprocess applications, and the possibility of mutations.     

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.     

Enumeration of airborne bacteria and fungi using solid phase cytometry

Conventional methods for the enumeration of airborne micro-organisms are inaccurate and time-consuming, hence the interest in novel approaches is increasing. In the present study, the use of solid phase cytometry (SPC) was evaluated for the enumeration of airborne micro-organisms. A 4 h SPC procedure based on viability staining was applied to samples from 50 locations and compared with an optimised culture-based method. Plate counts after air sampling were repeatable but strongly dependent on sampling volume. Samples with low or high microbial load were difficult to analyse using the culture-based method, unlike with SPC. Results show that SPC can be considered superior to the culture-based method because of its much higher dynamic range, its speed and its ability to enumerate not only culturable but all viable micro-organisms.     

Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone.

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.     

Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone.

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.     

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.     

Determination of Giardia cyst viability in environmental and faecal samples by immunofluorescence, fluorogenic dye staining and differential interference contrast microscopy

Amongst the techniques suggested for the determination of Giardia cyst viability, the use of the fluorogenic dyes, fluorescein diacetate (FDA) and propidium iodide (PI) is the most often recommended, even though it appears to overestimate the number of viable cysts. In the present study, the replacement of FDA with 4',6-diamidino-2-phenylindole (DAPI) allowed simultaneous direct immunofluorescence with monoclonal antibody labelled with fluorescein isothiocyanate (MAb-FITC). Under these conditions, it was possible both to quantify the cysts according to the immunofluorescence technique, and to appreciate their viability by using fluorogenic dye staining (DAPI and PI) and differential interference contrast (DIC) microscopy. This method proved to be significantly better than the counting methods normally suggested. The technique has been applied to Giardia cysts recovered from faeces and wastewater sludge.     

Maintenance and assessment of cell viability in formulation of non‐sporulating bacterial inoculants

The application of beneficial, plant‐associated microorganisms is a sustainable approach to improving crop performance in agriculture. However, microbial inoculants are often susceptible to prolonged periods of storage and deleterious environmental factors, which negatively impact their viability and ultimately limit efficacy in the field. This particularly concerns non‐sporulating bacteria. To overcome this challenge, the availability of protective formulations is crucial. Numerous parameters influence the viability of microbial cells, with drying procedures generally being among the most critical ones. Thus, technological advances to attenuate the desiccation stress imposed on living cells are key to successful formulation development. In this review, we discuss the core aspects important to consider when aiming at high cell viability of non‐sporulating bacteria to be applied as microbial inoculants in agriculture. We elaborate the suitability of commonly applied drying methods (freeze‐drying, vacuum‐drying, spray‐drying, fluidized bed‐drying, air‐drying) and potential measures to prevent cell damage from desiccation (externally applied protectants, stress pre‐conditioning, triggering of exopolysaccharide secretion, ‘helper’ strains). Furthermore, we point out methods for assessing bacterial viability, such as colony counting, spectrophotometry, microcalorimetry, flow cytometry and viability qPCR. Choosing appropriate technologies for maintenance of cell viability and evaluation thereof will render formulation development more efficient. This in turn will aid in utilizing the vast potential of promising, plant beneficial bacteria as sustainable alternatives to standard agrochemicals.     

The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci

AIMS: To investigate the ability of six fluorescent-based, two-colour viability assays to detect different physiological growth stages of two oral streptococci species. METHODS AND RESULTS: The growth of Streptococcus sanguinis and Strep. mutans from 0 to 73 h culture periods was monitored by cell labelling with six mixtures of fluorescent stains, in addition to the growth parameters optical density (O.D.), log values of the total cell counts (log BC ml(-1)) and of the colony-forming units (log cfu ml(-1)). CONCLUSION: In comparison with the corresponding cfu values as control, the vitality proportions determined by the Syto 9/PI test best reflected the dynamic growth pattern of both test strains. The direct fluorescent-based, two-colour assay Syto 9/PI provides valuable information about microbial viability stages. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of viable but non-culturable bacteria requires more precise direct methods such as the fluorescent staining technique presented here, in addition to the classical plate count method.     

Novel Method for Enumeration of Viable Lactobacillus plantarum WCFS1 Cells after Single-Droplet Drying

Survival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale.     

Preservation of micro-organisms by drying; a review

The preservation of micro-organisms by different drying methodologies has been used for decades. Freeze drying in particular is the preferred method for transporting and storing vast culture collections of micro-organism strain types. The literature on drying and preserving micro-organisms is extensive, but is often specific to one particular strain. This review attempts to draw some similar concepts and findings together in one paper, to compare different drying techniques, with specific reference to microorganisms. The main topics covered are cell growth phases and concentration, inducing drying tolerance in microbial cells, drying methods, rehydration of dried cells and packaging and storage conditions. Also, particular attention has been paid to the use of freeze drying and the protective matrices used to improve microbial cell viability after drying.     

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.     

Streamlined assessment of membrane permeability and its application to membrane engineering of Escherichia coli for octanoic acid tolerance

Journal of Industrial Microbiology & Biotechnology - The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for...     

A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting

To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K₂, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl₂ before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.     

Evidence of lethal and sublethal injury in food-borne bacterial pathogens exposed to high-intensity pulsed-plasma gas discharges

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to pulsed-plasma gas discharges (PPGD). METHODS AND RESULTS: The fluorescent redox probe 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used for enumerating actively respiring cells of Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica serovar Typhimurium that were suspended in sterile water at 4 degrees C and exposed to separate PPGD and heat treatments. While there was good agreement between use of respiratory staining (RS) and direct-selective agar plate counting (PC) for enumerating untreated bacteria, there were c. 1 and 3 log-unit differences in surviving cell numbers per millilitre for test organisms subjected to PPGD and heat treatments respectively, when enumerated by these different viability indicators. PPGD-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: Use of this RS method revealed that substantial subpopulations of test bacteria rendered incapable of forming colonies by separate PPGD and heat treatments may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this RS method offers interesting perspectives on assessing established and novel microbial inactivation methods, and may also provide a better understanding of mechanisms involved in microbial inactivation induced by high-intensity PPGD treatments.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Possible overestimation of surface disinfection efficiency by assessment methods based on liquid sampling procedures as demonstrated by in situ quantification of spore viability

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.     

Separation, Identification, and Characterization of Microorganisms by Capillary Electrophoresis

The use of capillary electrophoresis (CE) for the analysis, identification, and characterization of microorganisms has been gaining in popularity. The advantages of CE, such as small sample requirements, minimal sample preparation, rapid and simultaneous analysis, ease of quantitation and identification, and viability assessment, make it an attractive technique for the analysis of microbial analytes. As this instrumental method has evolved, higher peak efficiencies have been achieved by optimizing CE conditions, such as pH, ionic strength, and polymer additive concentration. Experimental improvements have allowed better quantitation and more accurate results. Many practical applications of this technique have been investigated. Viability and identification of microbes can be accomplished in a single analysis. This is useful for evaluation of microbial analytes in consumer products. Diagnosis of microbe-based diseases is now possible, in some cases, without the need for culture methods. Microbe-molecule, virus-antibody, or bacteria-antibiotic interactions can be monitored using CE, allowing for the screening of possible drug candidates. Fermentation can be monitored using this system. This instrumental approach can be adapted to many different applications, including assessing the viability of sperm cells. Progress has been made in the development of microelectrophoresis instrumentation. These advances will eventually allow the development of small, dedicated devices for the rapid, repetitive analyses of specific microbial samples. Although these methods may never fully replace traditional approaches, they are proving to be a valuable addition to the collection of techniques used to analyze, quantitate, and characterize microbes. This review outlines the recent developments in this rapidly growing field.