Search

Objectives Contrast-enhanced computed tomography (CECT) is used to monitor technical success immediately after hepatic microwave ablation (MWA). However, it remains unclear, if CECT shows the exact extend of the thermal destruction zone, or if tissue changes such as peri-lesionary edema are depicted as well. The objective of this study was to correlate immediate post-interventional CECT with histological and macroscopic findings in hepatic MWA in porcine liver in vivo. Methods Eleven MWA were performed in porcine liver in vivo with a microwave generator (928 MHz; energy input 24 kJ). CECT was performed post-interventionally. Livers were explanted and ablations were bisected immediately after ablation. Samples were histologically analyzed after vital staining (NADH-diaphorase). Ablation zones were histologically and macroscopically outlined. We correlated histologic findings, macroscopic images and CECT. Results Three ablation zones were identified in histological and macroscopic findings. Only one ablation zone could be depicted in CECT. Close conformity was observed between histological and macroscopic findings. The ablation zone depicted in CECT overestimated the histological avital central zone and inner red zone (p .05). Conclusions Immediate post-interventional CECT overestimated the clinically relevant zone of complete cell ablation after MWA in porcine liver in vivo. This entails the risk of incomplete tumor ablation and could lead to tumor recurrence.

Background More than 20% of the world’s agricultural land is affected by salinity, resulting in multibillion-dollar penalties and jeopardising food security. While the recent progress in molecular technologies has significantly advanced plant breeding for salinity stress tolerance, accurate plant phenotyping remains a bottleneck of many breeding programs. We have recently shown the existence of a strong causal link between salinity and oxidative stress tolerance in cereals (wheat and barley). Using the microelectrode ion flux estimation (MIFE) method, we have also found a major QTL conferring ROS control of ion flux in roots that coincided with the major QTL for the overall salinity stress tolerance. These findings open new (previously unexplored) prospects of improving salinity tolerance by pyramiding this trait alongside with other (traditional) mechanisms. Results In this work, two high-throughput phenotyping methods—viability assay and root growth assay—were tested and assessed as a viable alternative to the (technically complicated) MIFE method using barley as a check species. In viability staining experiments, a dose-dependent H2O2-triggered loss of root cell viability was observed, with salt sensitive varieties showing significantly more damage to root cells. In the root growth assays, relative root length (RRL) was measured in plants grown in paper rolls under different H2O2 concentrations. The biggest difference in RRL between contrasting varieties was observed for 1 mM H2O2 treatment. Under these conditions, a significant negative correlation in the reduction in RRL and the overall salinity tolerance is reported. Conclusions These findings offer plant breeders a convenient high throughput method to screen germplasm for oxidative stress tolerance, targeting root-based genes regulating ion homeostasis and thus conferring salinity stress tolerance in barley (and potentially other species). Electronic supplementary material The online version of this article (10.1186/s13007-019-0397-9) contains supplementary material, which is available to authorized users.

[EN] Available disinfection methods and therapies against Helicobacter pylori have multiple disadvantages, such as increased prevalence of antibiotic-resistant strains, which requires the search for novel effective antimicrobial agents against H. pylori. Among them, naturally-occurring antimicrobial compounds, like essential oil components (EOCs), have been reported as substances with anti-H. pylori potential. To avoid the disadvantages associated with using EOCs in their free form, including volatility, low water solubility and intense sensory properties, their immobilisation in inert supports has recently been developed. This study sought to evaluate the inhibitory properties of EOCs immobilised on silica microparticles against H. pylori and to elucidate the mechanism of action of the immobilised antimicrobials. After the preparation and characterisation of the antimicrobial supports, the susceptibility of H. pylori in the presence of the immobilised compounds was assessed by plate count, fluorescent viability staining and direct viable count-fluorescent in situ hybridisation analyses. The antimicrobial supports were found to inhibit H. pylori growth, and to induce morphological and metabolic alterations to the H. pylori membrane, with a minimum bactericidal concentration value between 25 and 50 mu g/ml according to the tested EOC. These findings indicate that immobilised EOCs can be used as potential antimicrobial agents for H. pylori clearance and treatment., Authors gratefully acknowledge the financial support from the Ministerio de Ciencia, Innovacion y Universidades, the Agencia Estatal de Investigacion and FEDER-EU (Project RTI2018-101599-B-C21). M.R.R. acknowledges the Generalitat Valenciana for their Postdoctoral Fellowship (APOSTD/2019/118).

Spore inoculum quality in filamentous bioprocesses is a critical parameter associated with viable spore concentration (1) and spore germination (2). It influences pellet morphology and, consequently, process performance. The state-of-the-art method to measure viable spore concentration is tedious, associated with significant inherent bias, and not applicable in real-time. Therefore, it is not usable as process analytical technology (PAT). Spore germination has so far been monitored using image analysis, which is hampered by complex medium background often observed in filamentous bioprocesses. The method presented here is based on the combination of viability staining and large-particle flow cytometry which enables measurements in real-time and hence aims to be applicable as a PAT tool. It is compatible with the complex media background and allows the quantification of metabolically active spores and the monitoring of spore germination. A distinction of germinated spores and not germinated spores was based on logistic regression, using multiparameteric data from flow cytometry. In a first step, a significant correlation between colony-forming unit (CFU) counts and viable spore concentration (1) in an industrially relevant model bioprocess was found. Spore germination (2) was followed over the initial process phase with close temporal resolution. The validation of the method showed an error below 5 %. Differences in spore germination for various spore inocula ages and spore inoculum concentrations were monitored. The real-time applicability of the method suggests the implementation as a PAT tool in filamentous bioprocesses. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7787-y) contains supplementary material, which is available to authorized users.

This study demonstrates a new technique for separating and purifying viable microbes from samples that interfere with viability staining. The viability ofBifidobacterium longumATCC 15707 was assessed using PBDC to separate bacteria from complex non-dairy food matrices and Quantitative Fluorescence Microscopy (QFM) to determine individual cells using LIVE/DEADBacLight bacterial viability staining. Water agar (3%) was used to retain cells ofB. longumand offered a lower fluorescence background withBacLight viability staining, compared with fixation on polycarbonate (PC) black membrane. The effect of drying temperatures and non-dairy foods on viability ofB. longumwas assessed.B. longumcoated on oat, peanut or raisin was separated by filtration, low- and high-speed centrifugation, flotation and sedimentation buoyant density centrifugation. Purified cells were subsequently deposited on water agar for rehydration followed by LIVE/DEADBacLight viability staining and enumeration. Conventional plate counting was also conducted to compare viability results. Finally, the applicability of this novel method for viability assessment was demonstrated and informative information of cell membrane damages ofB. longumincorporated onto non-dairy foods during 24 h drying was observed. Viability assessment ofB. longumcoated onto oat, peanut, or raisin was much lower by plate counting compared to viability staining. Drying appeared to have a greater impact when viability was assessed by plate counting compared to viability staining.IMPORTANCEEnumeration of viable beneficial bacteria from function foods presents a significant bottle neck for product development and quality control. Interference with microscopic and/or fluorescent techniques by ingredients, time required to incubate plated microbes, and the transient nature of the colony forming unit make rapid assessment of viable bacteria difficult. Viability assessment ofBifidobacterium longumATCC 15707 by Percoll Buoyant Density Gradient Centrifugation with LIVE/DEADBacLight viability staining on water agar (3%) was in agreement with serial dilution enumeration. Without the need for incubation viability assessment by staining provided a more rapid means to assess the impact of drying on the viability ofB. longumcoated onto oat, peanut or raisin.

Sperm viability (SV) is a widely applied measure of sperm quality but our review of the ecological literature revealed that previously identified methodological pitfalls have not been overcome, including low cross-study standardisation of protocols, inadequate statistical treatment, and unaccounted for within-sample heterogeneity. SV is additionally affected by biological variation such as between species, reproductive organs, or sperm age cohorts. Using SV per se as a surrogate for sperm quality may, therefore, be problematic. Sperm viability (SV), the proportion of live sperm in a sample, is a widely applied measure of sperm quality but few studies test its robustness. At least three reasons make SV problematic as a surrogate for sperm quality. First, reviewing the ecological literature revealed that previously identified methodological pitfalls have not been overcome, including low cross-study standardisation of protocols, inadequate statistical treatment, and unaccounted for within-sample heterogeneity. Second, SV is affected by biological variation such as between species, reproductive organs, or sperm age cohorts. Third, the proportion of live sperm extracted from males appears more related to male than to sperm quality in the sense of the future performance of sperm. We propose an alternative method to assess sperm quality by characterising the temporal decrease of SV in a stressor medium and illustrate in two species, the common bedbug (Cimex lectularius) and the fruit fly (Drosophila melanogaster) how some common methodological pitfalls may be circumvented. Our data empirically support the well-known but little-considered facts that i) non-blind measurements alter SV and ii) that SV frequently have non-significant repeatability within one sample. iii) Cross-sectional sampling of ejaculates showed that this heterogeneity even masked a biological pattern - the sperm stratification within males. We show iv) that this shortcoming can be overcome by following the temporal decline of SV of a sperm subsample in a stress test. To measure sperm quality, we advocate analysing the temporal decline in SV in a stressor medium over current protocols that use SV per se and blinding samples for SV measurements. As cell viability is widely used in biological and medical laboratory studies, our protocol may be useful to characterise cell quality beyond

To understand the response of the Lyme disease spirochete Borrelia burgdorferi exposed to stress conditions and assess the viability of this spirochete, we used a correlative cryo-fluorescence and cryo-scanning microscopy approach. This approach enables simple exposition of bacteria to various experimental conditions that can be stopped at certain time intervals by cryo-immobilization, examination of cell viability without necessity to maintain suitable culture conditions during viability assays, and visualization of structures in their native state at high magnification. We focused on rare and transient events e.g., the formation of round bodies and the presence of membranous blebs in spirochetes exposed to culture medium, host sera either without or with the bacteriolytic effect and water. We described all crucial steps of the workflow, particularly the influence of freeze-etching and accelerating voltage on the visualization of topography. With the help of newly designed cryo-transport device, we achieved greater reproducibility.

The production of an artisanal beer, made by brewers using traditional practices on a small scale, is founded on the empirical adjustment of parameters, including yeasts handling and serial repitching. The aim of this study was to monitor yeast viability during different stages of artisanal beer productions through the Live/Dead Yeast viability staining and to correlate it with fermentation dynamics in order to increase process standardization and to maintain the quality of final products. Yeast viability and fermentation activities were evaluated during seven fermentation cycles of an artisanal pilsner beer. Yeast inoculated with higher viability performed generally better in fermentation, resulting in faster sugar consumption, faster ethanol production and stability. Handling yeast and serial repitching based on Live/Dead viability measurements, could be the key way to ensure reliable manufacture of high quality beer and to improve process standardization particularly for microbreweries, where variability of production can be a challenging point.

It is difficult to determine whether early tuberculosis treatment is effective in reducing the infectiousness of patients' sputum, because culture takes weeks and conventional acid-fast sputum microscopy and molecular tests cannot differentiate live from dead tuberculosis.To assess treatment response, sputum samples (n=124) from unselected patients (n=35) with sputum microscopy-positive tuberculosis were tested pretreatment and after 3, 6, and 9 days of empiric first-line therapy. Tuberculosis quantitative viability microscopy with fluorescein diacetate, quantitative culture, and acid-fast auramine microscopy were all performed in triplicate.Tuberculosis quantitative viability microscopy predicted quantitative culture results such that 76% of results agreed within ±1 logarithm (rS=0.85; P.0001). In 31 patients with non-multidrug-resistant (MDR) tuberculosis, viability and quantitative culture results approximately halved (both 0.27 log reduction, P.001) daily. For patients with non-MDR tuberculosis and available data, by treatment day 9 there was a10-fold reduction in viability in 100% (24/24) of cases and quantitative culture in 95% (19/20) of cases. Four other patients subsequently found to have MDR tuberculosis had no significant changes in viability (P=.4) or quantitative culture (P=.6) results during early treatment. The change in viability and quantitative culture results during early treatment differed significantly between patients with non-MDR tuberculosis and those with MDR tuberculosis (both P.001). Acid-fast microscopy results changed little during early treatment, and this change was similar for non-MDR tuberculosis vs MDR tuberculosis (P=.6).Tuberculosis quantitative viability microscopy is a simple test that within 1 hour predicted quantitative culture results that became available weeks later, rapidly indicating whether patients were responding to tuberculosis therapy.

Determination of microbial viability by the plate count method is routine in microbiology laboratories worldwide. However, limitations of the technique, particularly with respect to environmental microorganisms, are widely recognized. Many alternatives based upon viability staining have been proposed, and these are often combined with techniques such as image analysis and flow cytometry. The plethora of choices, however, adds to confusion when selecting a method. Commercial staining kits aim to simplify the performance of microbial viability determination but often still need adaptation to the specific organism of interest and/or the instruments available to the researcher. This review explores the meaning of microbial viability and offers guidance in the selection and interpretation of viability testing methods.

To our knowledge the optimal freeze cycle length in renal cryotherapy is unknown. Ten-minute time based freeze cycles were compared to temperature based freeze cycles to -20C.Laparoscopic renal cryotherapy was performed on 16 swine. Time based trials consisted of a double 10-minute freeze separated by a 5-minute thaw. Temperature based trials were double cycles of 1, 5 or 10-minute freeze initiated after 1 of 4 sensors indicated -20C. A 5-minute active thaw was used between freeze cycles. Control trials consisted of cryoneedle placement for 25 minutes without freeze or thaw. Viability staining and histological analysis were done.There was no difference in cellular necrosis between any of the temperature based freeze cycles (p = 0.1). Time based freeze cycles showed more nuclear pyknosis, indicative of necrosis, than the 3 experimental freeze cycles for the renal cortex (p = 0.05) but not for the renal medulla (p = 0.61). Mean time to -20C for freeze cycle 1 was 19 minutes 10 seconds (range 9 to 46 minutes). In 4 of 21 trials (19%) -20C was never attained despite freezing for 25 to 63 minutes.There was no difference in immediate cellular necrosis among double 1, 5 or 10-minute freeze cycles. Cellular necrosis was evident on histological analysis for trials in which -20C was attained and in freeze cycles based on time alone. With a standard 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attained -20C. Cell death appeared to occur at temperatures warmer than -20C during renal cryotherapy.

The causes and consequences of variation in sperm quality, survival and ageing are active areas of research in ecology and evolution. In order to address these topics, many recent studies have measured sperm viability using fluorescent staining. Although sperm viability staining has produced a number of interesting results, it has some potential pitfalls that have rarely been discussed. In the present paper, I review the major findings of ecology and evolution studies employing sperm viability staining and outline the method’s principle limitations. The key problem is that the viability assay may itself kill sperm, which is likely to confound many common experimental designs in addition to producing artificially low estimates of sperm viability. I further suggest that sperm number should be routinely measured in sperm viability studies, as it may be an important but overlooked source of spurious results. I provide methodological advice on sperm viability staining aimed at minimising artefacts and producing robust conclusions, and discuss possible avenues for future research.

In this study, the bactericidal activity of antibacterial monomer MDPB (12-methacryloyloxydodecylpyridinium bromide) against Streptococcus mutans was tested by a rapid method for monitoring viability. To S. mutans culture containing fluorescence staining solution that distinguishes live from dead cells, MDPB was added at a concentration of 250, 100, 50, or 10 microg/ml. Bacterial cells were observed by fluorescence microscopy and the percentage of dead cells was calculated. After 10, 20, or 30 minutes' contact with MDPB, the live/dead ratio was measured by fluorometry and viable counts (CFU) determined by the conventional plating method. Viability staining revealed that MDPB exhibited significant bactericidal effects at 50 microg/ml or greater (ANOVA, Fisher's PLSD test), and complete killing of the cells at 250 microg/ml of MDPB was demonstrated in conjunction with a plating method. The staining method thus provided a sensitive means to determine loss of viability, and indicated the strong killing effects of MDPB on S. mutans.

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.

Summary NPR1-dependent salicylic acid signalling controls sodium entry into the roots while preventing potassium loss through depolarization-activated outward-rectifying potassium and ROS-activated non-selective cation channels during salt and oxidative stresses., The role of endogenous salicylic acid (SA) signalling cascades in plant responses to salt and oxidative stresses is unclear. Arabidopsis SA signalling mutants, namely npr1-5 (non-expresser of pathogenesis related gene1), which lacks NPR1-dependent SA signalling, and nudt7 (nudix hydrolase7), which has both constitutively expressed NPR1-dependent and NPR1-independent SA signalling pathways, were compared with the wild type (Col-0) during salt or oxidative stresses. Growth and viability staining showed that, compared with wild type, the npr1-5 mutant was sensitive to either salt or oxidative stress, whereas the nudt7 mutant was tolerant. Acute salt stress caused the strongest membrane potential depolarization, highest sodium and proton influx, and potassium loss from npr1-5 roots in comparison with the wild type and nudt7 mutant. Though salt stress-induced hydrogen peroxide production was lowest in the npr1-5 mutant, the reactive oxygen species (ROS) stress (induced by 1mM of hydroxyl-radical-generating copper-ascorbate mix, or either 1 or 10mM hydrogen peroxide) caused a higher potassium loss from the roots of the npr1-5 mutant than the wild type and nudt7 mutant. Long-term salt exposure resulted in the highest sodium and the lowest potassium concentration in the shoots of npr1-5 mutant in comparison with the wild type and nudt7 mutant. The above results demonstrate that NPR1-dependent SA signalling is pivotal to (i) controlling Na+ entry into the root tissue and its subsequent long-distance transport into the shoot, and (ii) preventing a potassium loss through depolarization-activated outward-rectifying potassium and ROS-activated non-selective cation channels. In conclusion, NPR1-dependent SA signalling is central to the salt and oxidative stress tolerance in Arabidopsis.

The aim of the study was to investigate the acclimation of precultivated acetatefed aerobic granular sludge to a toxic xenobiotic biodegradation. Establishment of p-nitrophenol (PNP) biodegradation in acetate-fed aerobic granular sludge and concomitant changes in the microstructure and bacterial community were determined. Rapid establishment of PNP utilization was observed in the granular sludge when fed with PNP as the sole carbon source. The specific PNP removal was 36-mg h -1 g -1 granular biomass at an initial PNP concentration of 50 mg L -1. The presence of PNP resulted in significant membrane damage in a subpopulation of the bacterial consortium, as shown by BacLight viability staining. This was coincided with a significant decrease in the culturable bacterial diversity of the granular biomass. PCR-DGGE analysis revealed a shift and decrease in number of bands during the establishment of PNP biodegradation. Scanning electron microscopy showed the dominance of rod-shaped bacteria in the PNP-utilizing microbial granules. Our results suggest that acetate-fed granular sludge could be quickly adapted for PNP biodegradation. � Springer Science+Business Media, LLC 2011.

This study examines the effect of erythromycin on activated sludge bacteria and the inhibition of the pollution removal in batch reactors treating urban wastewater. Results showed the importance of exposure time to erythromycin. Inhibition of nitrification and COD removal was measured during a 4 h period for erythromytcin concentrations higher than 1 mg/L. A 4 µg/L erythromycin concentration inhibited COD removal during a 20 h exposure time. The effect of erythromycin on nitrification was variable depending on the sludge origin. Erythromycin inhibited the specific nitrification rate with sludge from Nancy WWTP, but increased the nitrification rate at the other facility (Epinal WWTP). The cell lysis resulted in destruction of activated sludge flocs. Microscopic techniques (epifluorescence and confocal laser scanning microscopy (CLSM)), combined with a fluorescent viability indicator, allowed us to study erythromycin time-kill activity.Viability staining results showed a latency time before the lower antibiotic concentrations began to kill bacteria. This latency time could be related to antibiotic adsorption and diffusion into activated sludge flocs as well as the rate of bateria death. The effect of erythromycin according to the bacterial Gram type was investigated with 3-dimensional Confocal Laser Scanning Microscopy (CLSM) time-lapse imaging combined with a Gram and Viability staining. Gram+ bacteria had a higher mortality rate than the Gram- bacteria. This result suggests that antibiotic in wastewater could change the activated sludge bacteria composition according to their Gram type by selecting bacteria the less sensitive to the antibiotics, L'objectif de ce travail est de caractériser l'effet de l'érythromycine sur l'activité bactérienne et l'inhibition de l'épuration dans les réacteurs de traitement des eaux usées urbaines. L'étude a montré l'importance du temps d'exposition à l'érythromycine. L'inhibition de la nitrification et de l'épuration de la DCO a été mesurée sur une période de 4 h pour des concentrations supérieures à 1 mg/L d'érythromycine alors que l'inhibition de l'épuration pour une concentration d'érythromycine de 4 µg/L a été mesurée suite à 20 h d'exposition. L'origine de la liqueur mixte est également déterminante : l'érythromycine a inhibé la nitrification avec les boues de Nancy mais n'a pas inhibé la nitrification avec les boues et Épinal. Par contre, l'inhibition de l'épuration de la DCO a été mesurée pour les deux boues. Des analyses microscopiques ont montré qu'en présence d'érythromycine les flocs bactériens se fractionnaient suite aux lyses bactériennes. La microscopie en épifluorescence et la microscopie confocale combinées à des marqueurs de viabilité fluorescents ont permis de déterminer les vitesses de mortalité bactérienne. Ces études ont mis en évidence un temps de latence qui précède la mortalité des bactéries. Ce temps de latence pourrait être lié aux mécanismes d'adsorption de l'antibiotique et de diffusion dans les flocs, ainsi qu'à la vitesse biologique de mortalité. Enfin, un marquage simultané Gram et viabilité (microscopie confocale 3D au cours du temps) a montré que l'érythromycine pourrait modifier la composition bactérienne des boues en sélectionnant les bactéries les moins sensibles

Background Allografts from many tissue banks are carefully processed and stored with the goal of preserving chondrocyte viability. However, the importance of living chondrocytes for graft stability is unclear, in part because actual viabilities of individual allografts at the time of placement are seldom known. Hypotheses Cell yields from allograft and fresh cartilage differ significantly if chondrocyte viability in allografts is lower than indicated by fluorescence staining with conventional viability probes. In addition, transmission electron microscopy will show significant differences in the percentage of morphologically abnormal chondrocytes in allograft and fresh cartilage. Study Design Controlled laboratory study. Methods Fluorescence viability staining, chondrocyte yield, and chondrocyte characteristics were studied in 8 commercial osteochondral allografts (7 hemicondyles, 1 talus) and 4 freshly harvested cartilage samples from an adult distal femur (age, 46 years), from an adult talus (age, 51 years), and from an adult tibial plateau (age, 29 years) and from a juvenile distal tibia (age, 9 years). Selected fresh and allograft specimens were repeatedly frozen and thawed to deliberately kill chondrocytes by membrane disruption. The findings were analyzed to determine if allograft and fresh cartilage were significantly different with respect to each of the 3 different outcome measures. Results Although fluorescent staining indicated that approximately 75% of chondrocytes were viable (calcein AM—labeled) in allograft cartilage, counterstaining with 4,′6-diamidino-2-phenylindole showed that fewer than 30% contained identifiable nuclei. In contrast, 100% of cells labeled as viable contained nuclei in fresh cartilage. Killing chondrocytes by freeze-thawing before staining did not diminish calcein AM staining in allograft cartilage but caused a significant reduction in fresh cartilage. The average yield of chondrocytes from allograft cartilage was less than 200 000/100 mg tissue, significantly lower than in fresh cartilage, which averaged more than 1.5 million/100 mg tissue. The yield from freeze-thawed controls was less than 24 000/100 mg. Cell numbers increased after 7 days of culture in all cases except for chondrocytes from freeze-thawed cartilage, an indication that the isolated cells were viable. Morphologic analysis by transmission electron microscopy revealed significant increases in the numbers of chondrocytes with pyknotic or absent nuclei or with disintegrated plasma membranes in allograft versus fresh cartilage. Conclusion Conventional fluorescence probes are unreliable for analyzing chondrocyte viability in osteoarticular allografts. Alternative methods for assessment of viability, such as cell culture and ultrastructural imaging, may provide more accurate assessment of viability in allografts. Clinical Relevance Conventional staining methods that overestimate chondrocyte viability in osteoarticular allografts may mislead investigators attempting to assess the effects of chondrocyte viability on graft stability following implantation. A more reliable means to measure chondrocyte viability will be required to accurately assess these effects.

Viability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope. We found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome. When viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.