Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol.

The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population. A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented. The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater. The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts. The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.     

Increased growth yields of Mycoplasma spp. in the presence of pyruvate

Viable and non-viable African green monkey kidney (Vero) cells after treatment with Clostridium perfringens enterotoxin (CPE) followed by simultaneous double staining with fluorescein diacetate (FDA) and propidium iodide (PI) were counted with a flow cytometer (FCM). Within 1 min the FCM analysed 10 000 Vero cells in a sample for viability. After treatment of Vero cells with CPE for 60 min and staining with FDA-PI for 5 min, a reproducible dose-response curve was obtained between 25 and 400 ng/ml of CPE and percentage viable cell numbers. The FCM analysis proved to be a strong tool for rapid discrimination between viable and non-viable Vero cells treated with CPE in a large number of samples at a time.     

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.     

Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix

The interactions of DNA-binding dyes (Hoechst 33258, DAPI, acridine orange) and DiBAC₄(3) with hyperthermophilic archaeon Aeropyrum pernix cells were investigated by the combination of calorimetric, spectroscopic and microscopic techniques. All of the dyes, studied here, affect the thermal stability of DNA in vivo and in vitro. Hoechst 33258 is highly DNA-specific probe, which does not affect the thermal transitions of other cellular components as can be detected by differential scanning calorimetry (DSC). Due to this unique property, it can be used as a potential DNA marker for in vivo DSC studies. The localization of the dyes in the cells and viability assay was revealed by fluorescence microscopy. Hoechst 33258, DAPI and acridine orange did not distinguish between viable and non-viable cells of Aeropyrum pernix. Only with the commercially available Live/Dead BacLight^TM kit we were able to discriminate viable and non-viable Aeropyrum pernix cells.     

A new method for the rapid evaluation of gas vacuoles regeneration and viability of cyanobacteria by flow cytometry

Flow cytometric analysis for measuring gas vacuole regeneration and viability of cyanobacteria was developed. This novel approach distinguished between cyanobacteria with intact and collapsed gas vacuoles. By this method, sonicated cyanobacteria under illuminated conditions were shown to regenerate their gas vacuoles to the level of the untreated cells within 1–3 days. Ultrasonically treated cyanobacteria cultured under non-aerated and non-illuminated conditions did not regenerate their gas vacuoles. Combined with dual staining, viable and non-viable cyanobacteria are easily and rapidly quantified or enumerated by measuring the intensity of red fluorescence and green fluorescence. A high correlation was found between the numbers of viable and non-viable cyanobacteria with flow cytometric measurement.     

The use of fluorogenic esters to detect viable bacteria by flow cytometry

The ability of flow cytometry (FCM) to detect viable bacteria after staining with a range of fluorogenic esters was investigated with several bacterial species. The dyes studied were the fluorescein diacetate (FDA) derivatives carboxyfluorescein diacetate, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester and calcein acetoxymethyl ester, as well as ChemChrome B, a commercially-available stain for the detection of viable bacteria in suspension. No one dye was found to be universal but ChemChrome B dye stained the widest number of Gram-positive and Gram-negative species, whereas the FDA derivatives preferentially stained Gram-positive bacteria. The use of ChemChrome B to detect viable bacteria in environmental samples was investigated further by studying the survival of Klebsiella pneumoniae in lakewater. During survival studies, a higher number of viable bacteria were detected both by direct viable counts and FCM after staining with rhodamine 123 and ChemChrome B than by colony-forming units, suggesting the presence of viable but nonculturable cells. These results demonstrate the potential use of FCM to enumerate viable bacteria in natural waters.     

Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway

The viable and non-viable fractions of the bacterial community in a 2347-year-old permafrost soil from Spitsbergen were subjected to a comprehensive investigation using culture-independent and culture-dependent methods. LIVE/DEAD BacLight staining revealed that 26% of the total number of bacterial cells were viable. Quantitatively, aerobic microcolonies, aerobic colony-forming units and culturable anaerobic bacteria comprised a minor fraction of the total number of viable bacteria, which underlines the necessity for alternative cultivation approaches in bacterial cryobiology. Sulfate reduction was detected at temperatures between -2 degrees C and 29 degrees C while methanogenesis was not detected. Bacterial diversity was high with 162 operational taxonomic units observed from 800 16S rDNA clone sequences. The 158 pure cultures isolated from the permafrost soil affiliated with 29 different bacterial genera, the majority of which have not previously been isolated from permafrost habitats. Most of the strains isolated were affiliated to the genera Cellulomonas and Arthrobacter and several of the pure cultures were closely related to bacteria reported from other cryohabitats. Characterization of viable bacterial communities in permafrost soils is important as it will enable identification of functionally important groups together with the as yet undescribed adaptations that bacteria have evolved for surviving subzero temperatures for millennia.     

A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy

Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by ~25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80°C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies.     

Rapid assessment of bacterial viability by flow cytometry

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper     

Evaluation of ChemChrome V6 for bacterial viability assessment in waters

The efficiency of ChemChrome B (CB) and ChemChrome V6 (CV6) dyes to stain viable bacterial cells in water was compared. Both dyes are fluorogenic esters converted to free fluorescein by esterase activity. The dyes were applied to a wide variety of bacterial species, including those poorly stained by CB, and to natural waters. Some species tested gave unacceptable low fluorescence intensities by being inefficiently or non-labelled with the CB. In contrast, CV6-stained bacteria were easily detected by both flow cytometry and solid-phase cytometry. As a consequence, higher viable cell counts were found with CV6 compared with CB in natural waters. Viable counts determined by CV6 staining were always higher than cfu counts. In contrast, respiring cell counts (CTC) were always lower than CV6 counts and, in the case of tap and mineral waters, they were lower than cfu counts.     

Evaluation of Voltage-Sensitive Dyes for Long-Term Recording of Neural Activity in the Hippocampus

We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr. Received: 16 June 1999/Revised: 4 August 1999     

Reducing bias in bacterial community analysis of lower respiratory infections

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core'' and ‘satellite'' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.     

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.     

Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Even so, the lack of differentiation between DNA from viable and dead cells is one of the major challenges for diagnostic DNA-based methods. Certain nucleic acid-binding dyes can selectively enter dead bacteria and subsequently be covalently linked to DNA. Ethidium monoazide (EMA) is a DNA intercalating dye that enters bacteria with damaged membranes. This dye can be covalently linked to DNA by photoactivation. Our goal was to utilize the irreversible binding of photoactivated EMA to DNA to inhibit the PCR of DNA from dead bacteria. Quantitative 5'-nuclease PCR assays were used to measure the effect of EMA. The conclusion from the experiments was that EMA covalently bound to DNA inhibited the 5'-nuclease PCR. The maximum inhibition of PCR on pure DNA cross-linked with EMA gave a signal reduction of approximately -4.5 log units relative to untreated DNA. The viable/dead differentiation with the EMA method was evaluated through comparison with BacLight staining (microscopic examination) and plate counts. The EMA and BacLight methods gave corresponding results for all bacteria and conditions tested. Furthermore, we obtained a high correlation between plate counts and the EMA results for bacteria killed with ethanol, benzalkonium chloride (disinfectant), or exposure to 70 degrees C. However, for bacteria exposed to 100 degrees C, the number of viable cells recovered by plating was lower than the detection limit with the EMA method. In conclusion, the EMA method is promising for DNA-based differentiation between viable and dead bacteria.     

Enhanced immune response to pneumococcal infection in malnourished mice nasally treated with heat-killed Lactobacillus casei

The present study analyzed whether nasal administration of viable and non-viable Lactobacillus casei CRL 431 to immunocompromised mice was capable of increasing resistance against Streptococcus pneumoniae. Weaned mice were malnourished after consuming a PFD for 21 days. Malnourished mice were fed a BCD for 7 days or BCD for 7 days with viable or non-viable L. casei nasal treatments on day 6 and day 7 (BCD+LcV and BCD+LcN, respectively). The MNC group received PFD whereas the WNC mice consumed BCD. MNC mice showed greater lung colonization, more severe lung injuries, impaired leukocyte recruitment and reduced antibodies and cytokine production when compared with WNC mice. Administration of L. casei increased the resistance of malnourished mice to the infection. Both BCD+LcV and BCD+LcN treatments prevented the dissemination of the pathogen to the blood and induced its lung clearance. BCD+LcV or BCD+LcN groups showed improved production of TNF-α and activity of phagocytes in the respiratory tract, an effect that was not observed in the BCD control group. In addition, IL-4 and IL-10 were significantly increased in BCD+LcV and BCD+LcN groups, which correlated with the increase in the levels of specific respiratory IgA. The nasal treatments with L. casei were also effective at stimulating the production of specific IgG at both the systemic and the respiratory levels. The comparative study between the viable and the non-viable bacteria demonstrated that viability would be an important factor to achieve maximum protective effects. However, the results from this study suggest that heat-killed lactic acid bacteria are also effective in the immunomodulation of the systemic and respiratory immune system.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

Use of the direct epifluorescent filter technique for the enumeration of yeasts

The ability of the direct epifluorescent filter technique (DEFT) to enumerate the viable numbers of various species of yeasts was evaluated. A DEFT count could be made in less than 10 min and the DEFT counts of non-heat-treated samples agreed well with plate counts. The DEFT was unsuitable for the enumeration of yeasts in heat-treated samples because non-viable cells fluoresced orange. A double staining technique using Janus Green B and acridine orange was developed to overcome this problem. The modified DEFT enabled viable and non-viable yeasts to be differentiated in heat-treated samples of pure cultures and improved the relationship between the DEFT count and plate count. The method proved to be of little value, however, for use with beverage products because of unreliable staining patterns.     

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.     

Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry

BACKGROUND: Conventional techniques used to assess bactericidal activities of antibodies are time-consuming; flow cytometry has been used as a rapid alternative. In this study, the membrane potential-sensitive fluorescent probes bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) and Sytox Green, the redox dye cyano-2,3-ditolyl tetrazolium chloride (CTC), and the Baclite viability test kit were used to assess the effects of ceftazidime, ampicillin, and vancomycin on clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively. METHODS: Bacterial cultures were grown to early exponential phase, at which point the antibiotics were added at their breakpoint values, and incubation was allowed to continue. At timed intervals, samples were stained and flow cytometric analysis was performed on a Skatron Argus 100 arc-lamp based dual-parameter flow cytometer. RESULTS: All the dyes successfully identified antibiotic-induced damage in the three strains, although different fluorescence responses between the dyes were observed. DiBAC4(3) and Sytox Green overestimated numbers of nonviable bacteria relative to loss of viability as judged by plate counts. CTC, a measure of respiratory activity, revealed antibiotic-induced population heterogeneity illus trated by the development of several subpopulations. The "live" component of the viability kit identified two populations corresponding to viable and nonviable organisms, whereas the "dead" component only revealed single populations, the fluorescence intensity of which increased with antibiotic exposure. CONCLUSIONS: Flow cytometry provides a rapid and sensitive technique for the evaluation of the antibacterial activities of antibiotics. The use of a range of fluorophores specific for different cellular characteristics may be beneficial, bearing in mind the different fluorescence responses observed among the dyes used here.     

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 μg/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed. Received: 12 August 1998 / Accepted: 11 November 1998