Viability Probe Research Articles

Synthesis of F-18 labeled resazurin by direct electrophilic fluorination.

We present the synthesis and characterization of F18-labeled fluorinated derivatives of resazurin, a probe for cell viability. The compounds were prepared by direct fluorination of resazurin with diluted [F18]-F2 gas under acidic conditions. The fluorination occurs into the ortho-positions to the hydroxyl group producing various mono-, di-, and trifluorinated derivatives. The properties of the fluorinated resazurins are similar to the parent compound with the addition of fluorine leading to decreased pKa values and a bathochromic shift of the absorption maxima. The fluorinated resazurin derivatives can be used as probes for observation of cell viability in various cells, tissues and organs using a combination of positron emission tomography and direct optical imaging of Cerenkov luminescence.

Rapid and specific enumeration of viable Bifidobacteria in dairy products based on flow cytometry technology: A proof of concept study

A rapid and specific analytical method to enumerate viable Bifidobacteria in dairy products containing a mix of lactic acid bacteria was developed to proof of concept. A polyclonal antibody against Bifidobacterium animalis ssp. lactis (B. lactis) was used for specific detection by flow cytometry (FCM). The antibody, which targeted the cell wall of B. lactis, had a high labelling efficiency of 95.5 ± 1.9% of the population. A viability probe, 5,6-carboxyfluorescein diacetate, was combined with the antibody for real-time quantification of viable B. lactis. Enumeration results from FCM on four different commercial dairy products that contain B. lactis showed a good correlation with the standard culture-based method. Moreover, it provided a species-specific method to enumerate viable B. lactis within 2 h; the standard method counts the total cultivable Bifidobacteria genus on plates after 72 h. This study demonstrated that FCM could be a rapid solution for probiotic analysis in dairy products.

Fluorescence Technologies for Evaluating Male Gamete (Dys)Function

For many years, andrologists have sought ways of assessing sperm fertility, especially of new sires entering the breeding chain. As knowledge of the complex processes that enable sperm to fertilize eggs has increased, it has become clearer that quantitative estimation of the fertilizing potential of a sire or an ejaculate is actually unlikely ever to be fully realized. Here, we propose that a better approach is to identify substandard males and semen samples. During the past decades, the use of fluorescence technologies in biomedical science has burgeoned, with the development of very powerful instrumentation such as confocal microscopy and flow cytometers of ever-increasing capabilities together with a vast range of fluorochromes and fluorochrome conjugates. This technology has been applied to andrology but thus far in only a relatively simple way. In this review, we offer strategies for assessing a large range of sperm functions thought to be related to fertilizing ability over a temporal window rather than at a single time point. From such an assessment profile, sperm samples that over-respond or do not respond sufficiently could be identified, termed dysfunctional and rejected. We outline the rationales behind such tests, present information on new potentially useful fluorochromes and current flow cytometer models that would be suitable for the multicolour multifunctional tests we propose, and we offer suggestions as to how andrologists might design such multicolour tests for themselves.

Real time assays for quantifying cytotoxicity with single cell resolution.

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.

Identification of metal-tolerant organisms isolated from the Plankenburg River, Western Cape, South Africa

The ability of biofilms to resist pollutants makes them advantageous for use in bioremediation. The objective of this investigation was to isolate metal-tolerant micro-organisms from a site along the Plankenburg River. Microbial biofilms cultivated in multi-channelled flow cells were exposed to varying concentrations of aluminium (Al), iron (Fe), copper (Cu), manganese (Mn), nickel (Ni) and zinc (Zn), stained with the BacLightTM viability probe, visualised using epifluorescence microscopy and analysed using ScionImage. Exposure to the highest Al, Fe, Cu and Mn concentrations increased the percentages of dead cells. A difference in live and dead cells after exposure to varying Zn and Ni concentrations was not evident. When exposed to the lowest concentrations, no notable difference could be detected in comparison with the untreated control. Possible metal-tolerant micro-organisms were identified from the exposed flow cells using polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing, followed by ClustalX alignment and phylogenetic analysis. Phylogenetic analysis identified a variety of organisms, including Bacillus sp., Pseudomonas sp., Delftia tsuruhatensis strain A90, Kocuria kristinae strain 6J-5b, Comamonas testosteroni WDL7, Stenotrophomonas maltophilia strain 776, Staphylococcus sp. MOLA:313, Micrococcus sp. TPR14, Sphingomonas sp. 8b-1 and Microbacterium sp. PAO-12. Two major clusters could be distinguished based on their Gram-reactions.Keywords: BacLightTM viability probe, biofilms, phylogenetic analysis, river water, ScionImage

Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis

To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries. The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds. Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts. The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.

Single-walled carbon nanotubes promote rat vascular adventitial fibroblasts to transform into myofibroblasts by SM22-α expression

The aim of this study was to explore whether single-wall carbon nanotubes (SWCNTs) can be used as artery tissue-engineering materials by promoting vascular adventitial fibroblasts (VAFs) to transform into myofibroblasts (MFs) and to find the signal pathway involved in this process. VAFs were primary cultured and incubated with various doses of SWCNTs suspension (0, 0.8, 3.2, 12.5, 50, and 200 μg/mL). In the present study, we used three methods (MTT, WST-1, and WST-8) at the same time to detect the cell viability and immunofluorescence probe technology to investigate the effects of oxidative injury after VAFs incubated with SWCNTs. Immunocytochemical staining was used to detect SM22-α expression to confirm whether VAFs transformed into MFs. The protein levels were detected by western blotting. The results of immunocytochemical staining showed that SM22-α was expressed after incubation with 50 μg/mL SWCNTs for 96 hours, but with oxidative damage. The mRNA and protein levels of SM22-α, C-Jun N-terminal kinase, TGF-β1, and TGF-β receptor II in VAFs increased with the dose of SWCNTs. The expression of the p-Smad2/3 protein was upregulated while the Smad7 protein was significantly down-regulated. Smad4 was translocated to the nucleus to regulate SM22-α gene expression. In conclusion, SWCNTs promoted VAFs to transform into MFs with SM22-α expression by the C-Jun N-terminal kinase/Smads signal pathway at the early stage (48 hours) but weakened quickly. SWCNTs also promoted the transformation by the TGF-βl/Smads signal pathway at the advanced stage in a persistent manner. These results indicate that SWCNTs can possibly be used as artery tissue-engineering materials.

The effect of hyperosmolar stimuli and cyclophosphamide on the culture of normal rat urothelial cells in vitro.

Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells (RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively. The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively, 14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%, respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l HS had the most harmful effect based on the percentage of necrotic and apoptotic cells.

Some biological features of Candida albicans mutants for genes coding fungal proteins containing the CFEM domain

Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1) coding for putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM) are described. The deletion of these genes resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher cell surface hydrophobicity levels and an increased ability to bind to inert or biological substrates. Confocal scanning laser microscopy using concanavalin A-Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains formed thinner and more fragile biofilms. These apparently contained lower quantities of extracellular matrix material and less metabolically active cells than their parental strain counterpart, although the relative percentage of mycelial forms was similar in all cases. The cell surface of C. albicans strains harbouring deletions for genes coding CFEM-domain proteins appeared to be severely altered according to atomic force microscopy observations. Assessment of the relative gene expression within individual C. albicans cells revealed that CFEM-coding genes were upregulated in mycelium, although these genes were shown not to affect virulence in animal models. Overall, this study has demonstrated that CFEM domain protein-encoding genes are pleiotropic, influencing cell surface characteristics and biofilm formation.

Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome

Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction.

An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines

The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the existing legislation and the approval of the new European law regarding the production and importation of chemicals, known as REACH. This new legal framework supports the development of alternative methods to animal experimentation encouraging the improvement and/or design of new methodological strategies for the toxicological evaluation of chemical compounds. Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best agreement between obtaining relevant information about the mechanism of toxic action and the use of alternative methods. Following this trend, this work presents an integrated cellular strategy in order to know the toxicity and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The experimental procedures are performed in two steps. The first one involves the systematic analysis of the main cellular targets using proliferation, viability and morphological probes. The second step relies upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic action and the cellular response. The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.

The reactivity of neonatal rabbits to the mammary pheromone as a probe for viability

Newborn rabbits depend on a daily nursing interaction with the mother to gain milk and to survive. During this interaction, they localise and seize the nipples displaying a typical behaviour triggered by maternal odour cues. The mammary pheromone constitutes such a signal in domestic rabbits: it elicits sucking-related movements in more than 90% of the pups. However, some newborns remain unresponsive to the presentation of the pheromone, even pups apparently healthy and highly motivated to suck. The main goal of the present study was therefore to explore the link between the unresponsiveness of rabbit pups to the mammary pheromone and their growth and survival in breeding conditions. To that end, 293 newborns from 30 litters were tested for their head searching-oral grasping responses to the mammary pheromone on days 1 and 3, and their milk intake and mortality were followed up from days 1 to 21. It was hypothesised that unresponsive newborns would have subsequent difficulties in finding nipples, sucking and surviving. Early weight and success in milk intake were further considered as mediating factors in growth and viability. The results showed that pups that were unresponsive to the mammary pheromone on day 1 were less successful in gaining milk and had a higher rate of mortality than the responsive pups. However, this impact was modulated by the weight of pups: it appeared only in the lightest newborns. Moreover, this impact vanished on day 3. On the other hand, the pup weight and sucking success on days 1 to 3 strongly influenced viability and growth during the period extending from days 1 to 21. Taken together, the results show that the day-1 responsiveness of rabbit pups to the mammary pheromone can be considered as an indicator of individual viability in pups having a small weight (<48 g on day 1). The predictive validity of the pups’ pheromonal reactivity seems however time-limited as it works only during the first, but crucial, postnatal days.

Levels of circulating endothelial cells (CECs) as early pharmacodynamic markers of activity of anti-angiogenic (AA) therapy in advanced pancreatic cancer (PC)

14080 Background: Clinical evaluation of targeted AA therapy may be enhanced by non-invasive biomarkers assessing relevant drug action. In this study we tested the hypothesis that AA therapy with bevacizumab (BV), may result in early release of mature CECs from the tumor vasculature into the peripheral circulation and that this change may serve as a surrogate pharmacodynamic marker of response. Methods: In an ongoing phase II study, patients with advanced PC received BV (15 mg/kg IV, d1), capecitabine (650 mg/m2 twice daily × 14 days) and gemcitabine (1000 mg/m2 IV d1 &amp; 8) given q21 days as first line therapy. CECs (CD34+, CD31+, and CD45− cells) were assessed pre treatment on d1 and on d3 of cycle 1 by 4-color flow cytometry. Quality of life (QOL) was assessed prior to each cycle using the PAN26 questionnaire. Radiographic response by RECIST criteria and CA 19–9 were assessed every 8 weeks. Kendall’s tau, the Wilcoxon signed rank test and Mann-Whitney U-test were used to investigate an association between biomarkers (CEC and CA 19–9), paired data and independent data, respectively. Results: Twenty six of planned 34 pts have been accrued; 13 pts are assessable for change in CECs, 10 are evaluable for response, 3 are too early. Patient demographics: M/F: 4/9; median age 63 years (range 38–79). Best response in 10 pts: 2 PR, 7 SD and 1 PD. On day 3, CECs increased in 8 and decreased in 5 pts compared to baseline. During treatment CA19–9 decreased in 7 and increased in 6 pts. No significant association between CEC and CA 19–9 was detected (Kendall’s tau=-0.2, p=0.367). No significant differences of CEC levels or CA 19–9 were found pre and post-treatment, QOL improved and unimproved, and response and no-response groups. Conclusion: Preliminary analysis in 10 patients reveals no correlation between change in CECs and QOL or radiographic or tumor marker response. This may be due to delayed change (beyond day 3) or release of non-viable CECs that are not currently measured. The limited sample at this early analysis may also account for this finding. These results will be updated as accrual continues with refined CEC measurements which include a viability probe. This study was supported by a grant from NCCN and Genentec pharmaceuticals No significant financial relationships to disclose.

A death round affecting a young compartmentalized mycelium precedes aerial mycelium dismantling in confluent surface cultures of Streptomyces antibioticus

Development-associated cell-death processes were investigated in detail during the growth and differentiation of Streptomyces antibioticus ATCC 11891 on confluent surface cultures, by using fluorescent viability probes, membrane and activity fluorescence indicators, and electron microscopy analysis. A previously unsuspected complexity was revealed, namely the presence of a very young compartmentalized mycelium that dies following an orderly pattern, leaving alternating live and dead segments in the same hypha. This death round is followed by the growth of a second mycelium which develops rapidly from the live segments of the first mycelium and dies massively in a second death round, which extends over the phases of aerial mycelium formation and sporulation.

Elevated Microbial Tolerance to Metals and Antibiotics in Metal-Contaminated Industrial Environments

To test the hypothesis that industrial metal contaminants select for microorganisms tolerant to unrelated agents, such as antibiotics, we analyzed metal and antibiotic tolerance patterns in microbial communities in the intake and discharge of ash settling basins (ASBs) of three coal-fired power plants. High-throughput flow-cytometric analyses using cell viability probes were employed to determine tolerances of entire bacterioplankton communities, avoiding bias toward culturable versus nonculturable bacteria. We found that bacterioplankton collected in ASB discharges were significantly more tolerant to metal and antibiotic exposures than bacterioplankton collected in ASB intakes. Optical properties of microorganisms collected in ASB discharges indicated no defensive physiological adaptations such as formation of resting stages or excessive production of exopolymers. Thus, it is likely that the elevated frequency of metal and antibiotic tolerances in bacterioplankton in ASB discharges were caused by shifts in microbial community composition, resulting from the selective pressure imposed by elevated metal concentrations or organic toxicants present in ASBs.

Distinct Signaling Pathways Are Involved in Leukosialin (CD43) Down-regulation, Membrane Blebbing, and Phospholipid Scrambling during Neutrophil Apoptosis

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 microM microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

A versatile assay for the accurate, time-resolved determination of cellular viability

A convenient and versatile method for the accurate, time-resolved determination of cellular viability has been developed. The conventional viability indicator fluorescein diacetate (FDA), which is converted to the fluorescent compound fluorescein in living cells, was employed as a viability probe. Fluorescence emission from cells was measured using a spectrofluorimeter equipped with a magnetic stirrer. Using this assay cell suspensions exhibiting densities in the range 0.5×10 5 to 2.0×10 5 cells displayed a linear response when FDA concentrations less than 12 μM were employed. To calibrate the method, viability standards were elaborated using different proportions of living and dead cells, and a correlation coefficient for the viability of tobacco BY-2 suspensions was calculated as 0.998. This viability assay was also found to be applicable to Chlamydomonas reinhardtii and Arabidopsis thaliana cultured cells. Using this cell viability assay, kinetic analyses of cell death could be performed. Using the proteinaceous elicitor from Phytophthora cryptogea, cryptogein, to induce cell death in tobacco cell suspensions, values for the maximum velocity of death induction rate ( V max) and the LD50 (half-maximal velocity or k 1/2) were calculated as 17.2 (% death/h) and 65 nM, respectively.

Pseudomonas aeruginosa biofilms react with and precipitate toxic soluble gold.

The interaction between biofilms of Pseudomonas aeruginosa PAO1 and 0.01-5 mM gold chloride was investigated using flow-cells. Scanning confocal laser microscopy (SCLM) of these biofilms revealed the formation of two distinct structural features: (i) confluent areas of uniform thickness and (ii) cell clusters which often emerged as 30-40 micro m, tall narrow pillars (or pedestals) of cells and exopolymeric substance (EPS). When 5-day-old, quasi-steady state biofilms (as indicated by the stability of film thickness and overall structure) were exposed to relatively high AuCl3 (i.e. 0.5-5 mM) for 30 min at 20 degrees C, reduction of the auric ion resulted in the formation of both extracellular and intracellular metallic gold colloids, as revealed by transmission electron microscopy (TEM). Most mineralization occurred on cell surfaces with lesser amounts within cells and little throughout the EPS. Little to no mineralization of gold was seen at 0.01-0.1 mM concentrations. As initial AuCl3 concentrations approached 0.5 mM or greater, more gold particles were seen and cell viability, as determined by a BacLight live/dead viability probe, approached zero. At an intermediate concentration of 0.1 mM, the live:dead ratio increased to 4:1. However, when planktonic cells were exposed to this same 0.1 mM concentration, it resulted in a 4-log reduction in viable counts as determined by plating. The higher resistance of biofilm cells to 0.1 mM gold can be attributed to its binding to the EPS and cell surfaces of the biofilm which ensured a (presumably) low effective cytoplasmic concentration of gold (i.e. no gold crystals were seen in cells by TEM). In addition, SCLM revealed the formation of larger extracellular gold crystals at the substratum (coverslip) level of the biofilms, with a higher proportion of crystals detected beneath pillars (cell cluster structures), suggesting the possibility of unique cell types, more reduced microenvironments at the base of each cluster, or a combination of both. These results suggest that the biomineralization of gold is impacted by biofilm structure.

Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters.

Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) and TOTO-1 [1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3dihydro(benzo-1,3-oxazole)-2-methylidene]-1-(3'-trimethylammoniumpropyl)-pyridinium tetraiodide] for (stressed) lactic acid bacteria (C. J. Bunthof, K. Bloemen, P. Breeuwer, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 67:2326-2335, 2001). cFDA stains intact cells with enzymatic activity, and TOTO-1 stains membrane-permeabilized cells. Here we used this assay to study the viability of bacterial suspensions in milk, dairy fermentation starters, and probiotic products. To facilitate FCM analysis of bacteria in milk, a commercially available milk-clearing solution was used. The procedure was optimized to increase the signal-to-noise ratio. FCM enumerations were accurate down to a concentration of 10(5) cells ml(-1). The level of retrieval of Lactobacillus plantarum WCFS 1 suspended in milk was high, and viability was not affected by the procedure. The plate counts for cleared samples of untreated cell suspensions were nearly as high as the total FCM counts, and the correlation was strong (r > 0.99). In dairy fermentation starters and in probiotic products the FCM total cell counts were substantially higher than the numbers of CFU. Three functional populations could be distinguished: culturable cells, cells that are intact and metabolically active but not culturable, and permeabilized cells. The proportions of the populations differed in the products tested. This FCM method provides tools to assess the functionality of different populations in fermentation starters and probiotic products.