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Phytophthora are eukaryotic microbes that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via homothallic or heterothallic reproduction. These resting spores can survive in the soil for several years in the absence of a host plant, thus providing a long-term inoculum for disease. The ability to quantitatively evaluate oospore viability is an important part of many phytopathology studies. Here, we tested six fluorescent viability dyes for their ability to differentially stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate (FDA), 5-carboxyfluorescein diacetate (CFDA), propidium iodide, and TOTO-3 iodide. Each dye was first tested individually with untreated or heat-treated oospores as proxies for viable and nonviable oospores, respectively. SYTO9, FUN-1, CFDA and propidium iodide stained untreated and heat-treated oospores indiscriminately. In contrast, FDA (a green-fluorescent viable cell stain) and TOTO-3 (a red-fluorescent nonviable cell stain) differentially stained untreated or heat-treated oospores with no cross-fluorescence. We then tested the efficacy of dual-viability staining in conjunction with a pipeline for automated image analysis. To validate the method, untreated and heat-treated oospores were mixed at specific ratios, dual-stained, and analyzed using the pipeline. Linear regression of the resulting data showed a clear correlation between the expected and measured oospore ratios (dy/dx = 0.95, R2 = 0.88). Overall, the combination of dual-fluorescence staining and automated image analysis provides a high-throughput method for quantitatively assessing oospore viability and therefore can facilitate further studies on this key part of the Phytophthora disease cycle. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The aim was to evaluate the antibacterial efficacy and penetration depth into dentinal tubules of a solution of chitosan nanodroplets (NDs) loaded with Benzalkonium Chloride (BAK). Seventy-two human single-root teeth with fully formed apex were used. Cylindrical root dentin blocks were longitudinally sectioned and enlarged to a size of a Gates Glidden drill #4. After sterilization, root canals were infected with Enterococcus faecalis ATCC 29212 and further incubated for three weeks. Specimens were assigned to three experimental groups (n = 20), plus positive (n = 6) and negative (n = 6) controls. In the first group, irrigation was achieved with 2 mL of NDs solution loaded with BAK (NDs-BAK), in the second with 2 mL of 5% sodium hypochlorite (NaOCl) and in the last with 2 mL of 2% chlorhexidine (CHX). Specimens were rinsed and vertically fractured. Confocal laser scanning microscopy (CLSM) and viability staining were used to analyze the proportions of dead and live bacteria quantitatively. The volume ratio of red fluorescence (dead) was calculated in 3D reconstructions. Data were analyzed by one-way ANOVA and post hoc Bonferroni tests (p < 0.05). The ratio of red fluorescence over the whole green/red fluorescence resulted in a significant comparison of NDs-BAK with NaOCl (p < 0.01) and NaOCl with CHX (p < 0.01). No differences were found between NDs-BAK and CHX (p > 0.05). The mean depth of efficacy was, respectively: NDs-BAK 325.25 μm, NaOCl 273.36 μm and CHX 246.78 μm with no statistical differences between groups. The NaOCl solution showed the highest antimicrobial efficacy, but nanodroplets with BAK seemed to have the same effect as CHX with a high depth of efficacy. [ABSTRACT FROM AUTHOR]

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.

Abstract 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).

The aim was to evaluate the antibacterial efficacy and penetration depth into dentinal tubules of a solution of chitosan nanodroplets (NDs) loaded with Benzalkonium Chloride (BAK). Seventy-two human single-root teeth with fully formed apex were used. Cylindrical root dentin blocks were longitudinally sectioned and enlarged to a size of a Gates Glidden drill #4. After sterilization, root canals were infected with Enterococcus faecalis ATCC 29212 and further incubated for three weeks. Specimens were assigned to three experimental groups (n = 20), plus positive (n = 6) and negative (n = 6) controls. In the first group, irrigation was achieved with 2 mL of NDs solution loaded with BAK (NDs-BAK), in the second with 2 mL of 5% sodium hypochlorite (NaOCl) and in the last with 2 mL of 2% chlorhexidine (CHX). Specimens were rinsed and vertically fractured. Confocal laser scanning microscopy (CLSM) and viability staining were used to analyze the proportions of dead and live bacteria quantitatively. The volume ratio of red fluorescence (dead) was calculated in 3D reconstructions. Data were analyzed by one-way ANOVA and post hoc Bonferroni tests (p < 0.05). The ratio of red fluorescence over the whole green/red fluorescence resulted in a significant comparison of NDs-BAK with NaOCl (p < 0.01) and NaOCl with CHX (p < 0.01). No differences were found between NDs-BAK and CHX (p > 0.05). The mean depth of efficacy was, respectively: NDs-BAK 325.25 μm, NaOCl 273.36 μm and CHX 246.78 μm with no statistical differences between groups. The NaOCl solution showed the highest antimicrobial efficacy, but nanodroplets with BAK seemed to have the same effect as CHX with a high depth of efficacy.

BACKGROUND: Animal liver is established as an ex vivo model for studies on hepatic microwave ablation (MWA). Macroscopically visible color changes in the ablation zone are used to assess cell destruction and confirm successful ablation ex vivo. OBJECTIVE: Macroscopy and histology of MWA zones regarding cell viability in ex vivo porcine livers were compared in this study. METHODS: Six MWA were performed in porcine livers post mortem. A 14-G antenna and microwave generator (928 MHz; 9.0 kJ) were used. MWA were cut at the maximum cross section in vertical alignment to the antenna. NADH-diaphorase staining determined cell vitality. Macroscopic and microscopic ablation zones were statistically analyzed. RESULTS: Histology showed two distinct ablation zones: central white zone (WZH) with no cell viability and peripheral red zone (RZH) with partial cell viability. However, the macroscopically visible WZM was significantly smaller than the microscopic WZH with an area difference of 43.1% (p < 0.05) and a radius difference of 21.2% (1.6 mm; p < 0.05). Macroscopy and histology showed a very high correlation for the complete lesion area (WZH/M+RZH/M; r = 0.9; p = 0.001). CONCLUSIONS: The avital central zone is significantly larger as the macroscopically visible WZ which is commonly used to assess successful ablation in MWA ex vivo studies. Irreversible cell destruction can be underestimated in macroscopic evaluation. [ABSTRACT FROM AUTHOR]

• Rapid isolation of bacteria using "bacteria-sized" gold-coated magnetic microdiscs. • Isolation of E. coli from up to 100 mL water samples (LOD: 102 CFU/100 mL). • Platform enables viability assay on the captured discs using fluorescent markers. • Application on E. coli detection in agricultural water (no enrichment, <45 min). This work introduces a method for rapid isolation of target bacterial cells using "bacteria-sized," gold-coated magnetic microdiscs (1.5 μm in diameter, 80 nm in thickness). These microdiscs are functionalized with DNA aptamers for selectively isolating Escherichia coli cells from lab-prepared and environmental water samples at concentrations as low as 102 CFU/100 mL with no enrichment. This microdisc platform enables viability assay on the magnetically captured discs using fluorescent markers such as SYTO9, propidium iodide, and carbon quantum dots, providing both a detection and viability step in a single analysis. The significance of this gravity-driven separation technique is the ability to rapidly isolate target cells from relatively large sample volumes (up to 100 mL) with simple field-deployable apparatus in one step, without the use of complicated equipment or energy. This technique has immediate application for rapid (< 45 min) isolation and detection of fecal indicators (E. coli) in agricultural water and can be used for drinking water analysis if an enrichment step is added. In addition to targeting E. coli , the platform can be used for targeting other cells based on functionalization of the microdiscs with other bioreceptors such as aptamers, lectins, phages, or peptides. [ABSTRACT FROM AUTHOR]

Oxygen depletion under waterlogged conditions results in a compromised operation of H+-ATPase, with strong implications for membrane potential maintenance, cytosolic pH homeostasis, and transport of all nutrients across membranes. The above effects, however, are highly tissue specific and time dependent, and the causal link between hypoxia-induced changes to the cell's ionome and plant adaptive responses to hypoxia is not well established. This work aimed to fill this gap and investigate the effects of oxygen deprivation on K+ signalling and homeostasis in plants, and potential roles of GORK (depolarization-activated outward-rectifying potassium) channels in adaptation to oxygen-deprived conditions in barley. A significant K+ loss was observed in roots exposed to hypoxic conditions; this loss correlated with the cell's viability. Stress-induced K+ loss was stronger in the root apex immediately after stress onset, but became more pronounced in the root base as the stress progressed. The amount of K+ in shoots of plants grown in waterlogged soil correlated strongly with K+ flux under hypoxia measured in laboratory experiments. Hypoxia induced membrane depolarization; the severity of this depolarization was less pronounced in the tolerant group of cultivars. The expression of GORK was down-regulated by 1.5-fold in mature root but it was up-regulated by 10-fold in the apex after 48 h hypoxia stress. Taken together, our results suggest that the GORK channel plays a central role in K+ retention and signalling under hypoxia stress, and measuring hypoxia-induced K+ fluxes from the mature root zone may be used as a physiological marker to select waterlogging-tolerant varieties in breeding programmes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Continuing research ex vivo and in vivo with animal models is performed to advance the oncological safety of radiofrequency ablation (RFA) of liver tumors. In these experiments, frequently imaging modalities (e.g. MRI or CT) or macro-morphological measurements are used to determine the full extent of the different ablation zones inside of RFA lesions. However, no systematic study has been performed so far, which verified the accuracy of the macro-morphological findings. Therefore, the present study aimed to correlate histological and gross pathological findings of bipolar radiofrequency ablation zones of porcine livers with regard to cell viability in vivo. METHODS: Bipolar RFA was performed in the liver of anaesthetized female domestic pigs under CT-guidance using an internally cooled 20 mm RFA applicator. Afterwards RFA cross sections of the liver were made in a perpendicular orientation to the applicator. Ablation zones were initially documented by photography and thereafter prepared for histological analysis. Latter was based on HE-staining and NADH-diaphorase cell viability staining. Micro- and macro-morphological sections were digitally analyzed along the cross-section area for statistical correlation. RESULTS: Three different RF ablation zones could be differentiated. A central zone showing no cell viability (white zone) was surrounded by a red zone. The red zone could be divided into an inner zone of viable and non-viable cells (red zone 1), followed by a zone of edema with mostly viable cells (red zone 2). Micro- and macro-morphological data showed a strong correlation for the white zone (r = 0.95,p<0.01), the red zone 1 (r = 0.85, p< 0.01), and the red zone 2 (r = 0.89, p< 0.01). CONCLUSION: White zone and red zone could clearly be distinguished in gross pathology and histology after bipolar RFA of porcine liver tissue in vivo. The red zone could be differentiated into an inner zone of viable and non-viable cells and an outer zone with high cell viability and intercellular edema. A strong correlation of micro- and macro-morphology could be shown for all three ablation zones. With this knowledge, gross pathological examination can be used as a reliable indicator of lethally damaged tissue in bipolar RFA of in vivo porcine liver. [ABSTRACT FROM AUTHOR]

Spore inoculum quality in filamentous bioprocesses is a critical parameter influencing pellet morphology and, consequently, process performance. It is essential to determine the concentration of viable spores before inoculation, to implement quality control and decrease batch-to-batch variability. The ability to assess the spore physiologic status with close-to-real time resolution would offer interesting perspectives enhanced process analytical technology (PAT) and quality by design (QbD) strategies. Up to now, the parameters contributing to spore inoculum quality are not clearly defined. The state-of-the-art method to investigate this variable is colony-forming unit (CFU) determination, which assesses the number of growing spores. This procedure is tedious, associated with significant inherent bias, and not applicable in real time. Here, a novel method is presented, based on the combination of viability staining (propidium iodide and fluorescein diacetate) and large-particle flow cytometry. It is compatible with the complex medium background often observed in filamentous bioprocesses and allows for a classification of the spores into different subpopulations. Next to viable spores with intact growth potential, dormant or inactive as well as physiologically compromised cells are accurately determined. Hence, a more holistic few on spore inoculum quality and early-phase biomass composition is provided, offering enhanced information content. In an industrially relevant model bioprocess, good correlation to CFU counts was found. Morphological parameters (e.g. spore swelling) that are not accessible via standard monitoring tools were followed over the initial process phase with close temporal resolution. [ABSTRACT FROM AUTHOR]

In stirred-tank photobioreactors agitation causes fragmentation of filamentous cyanobacteria. Here, we introduce a flow cytometric approach for high-throughput measurements of trichome dimensions, heterocysts and metabolic activity of Anabaena sp. cultures. The longest characterized trichome had 1135 μm chain length. This technology could potentially be used for monitoring and control purposes. • High-throughput method for morphological characterization of filamentous cyanobacteria • Physiological characterization via fluorescence staining methods • Potential on-line process analytical technology for photobioreactor cultivations [ABSTRACT FROM AUTHOR]

This chapter provides a protocol for a detailed evaluation of phytoplankton and nuisance cyanobacteria with the FlowCam 8400 and the FlowCam Cyano. The chapter includes (i) detailed description of the quality control of fluorescent mode of the FlowCam, (ii) detailing methods for discriminating nuisance cyanobacteria using the FlowCam Cyano, how to set up libraries and classification routines for commonly used classification reports, and (iii) detailing methods for viability staining to quantify LIVE versus DEAD phytoplankton using the FlowCam 8400., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

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 1 mM of hydroxyl-radical-generating copper-ascorbate mix, or either 1 or 10 mM 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. [ABSTRACT FROM AUTHOR]

* Premise of the study: Assessing seed quality in orchids has been hindered by stringent germination requirements. Seed quality has traditionally been assessed in orchids using in vitro or in situ germination protocols or viability staining. However, these methods are not always well suited for rapid assessment of viability in the context of ecological studies. * Methods: The potential of an ex situ protocol for seed viability assessment of orchids in ecological studies was investigated by sowing seeds of Platanthera blephariglottis on Sphagnum moss collected in the orchid's natural habitat. Ex situ germination results were compared with those obtained by viability staining using triphenyltetrazolium chloride (TTC), and the effect of seed testa color on staining and germination results was investigated. * Key results: The ex situ protocol yielded high germination rates, with 66% of the seeds germinating after 9 wk. Depending on the seed testa color class, ex situ germination rates were about 1.4 to 2.5 times higher than viability rates determined using TTC, indicating that the TTC technique underestimated viability compared with the method using ex situ germination. The TTC estimates of viability rates were higher for seeds with dark-colored testae than for pale ones, whereas seed testa color had no effect on germination. * Conclusions: Our study showed promising results for the use of ex situ germination as an alternative to previously developed protocols for seed viability assessment of orchids in ecological studies. Staining using TTC might not be well suited for this purpose, since it introduced a bias with respect to seed testa color. [ABSTRACT FROM AUTHOR]

Background: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Aims The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli. Methods and Results Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L. innocua and E. coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable ( VBNC) state after the pulsed light treatment. A high percentage of L. innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E. coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species ( ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH- DA and RAPD- PCR, respectively. Conclusions A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample−1. Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes. Significance and Impact of the Study The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment. [ABSTRACT FROM AUTHOR]

Pulsed electric fields (PEF) are known to be effective at permeabilizing plant tissues. Prior research has demonstrated that lower pulse frequencies induce higher rates of permeabilization, but the underlying reason for this response is unclear. Intriguingly, recent microscopic observations with onion tissues have also revealed a correlation between PEF frequency and the subsequent speed of intracellular convective motion, i.e., cytoplasmic streaming. In this paper, we investigate the effect of cytoplasmic streaming on the efficacy of plant tissue permeabilization via PEF. Onion tissue samples were treated with Cytochalasin B, a known inhibitor of cytoplasmic streaming, and changes in cellular integrity and viability were measured over a wide range of frequencies and field strengths. We find that at low frequencies (f < 1 Hz), the absence of cytoplasmic streaming results in a 19% decrease in the conductivity disintegration index compared with control samples. Qualitatively, similar results were observed using a microscopic cell viability assay. The results suggest that at low frequencies convection plays a statistically significant role in distributing more conductive fluid throughout the tissue, making subsequent pulses more efficacious. The key practical implication is that PEF pretreatment at low frequency can increase the rate of tissue permeabilization in dehydration or extraction processes, and that the treatment will be most effective when cytoplasmic streaming is most active, i.e., with freshly prepared plant tissues. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 [ABSTRACT FROM AUTHOR]

Object: Histology is the gold standard for confirming thermally induced necrosis. Generally, however, no specimen is obtained from thermal ablation therapy for pathological examination. The aim of this study was to provide evidence for the relationship between temperatures reached and resulting tissue coagulation during laser ablation in a near-physiological ex vivo lung tumor model by combining viability staining and direct temperature measurement. Materials and methods: In all, 17 human lung specimens with primary non-small-cell lung cancer (NSCLC) were examined in this study. Organs were resected with curative intent from patients of either gender (5 female, 12 male) with an average age of 65 years (51-78). Here, 11/17 specimens were subjected to interstitial laser thermal ablation in an ex vivo lung perfusion and ventilation model after surgery. A control group of 6/17 specimens was tested for viability without laser ablation. Tissue temperature was measured invasively in real-time during the ablation process using thermocouples. Afterwards, representative slices of all 17 specimens were tested for viability with triphenyltetrazolium chloride (TTC). Maximum tissue temperature Tmax[°C] measured at a distance of 10 and 20 mm from the laser tip and time of temperature exposure were correlated with the diameter of the induced coagulation as ascertained with viability staining. CH evaluated the results. Results: Mean maximum temperature was 75.9°C ± 14.4°C at a distance of 10 mm from the laser tip and 50.3°C ± 14.6°C at a distance of 20 mm, respectively. The mean distance between the coagulation margin and the laser tip was 17.8 mm ± 7.3 mm. Conclusion: We found that coagulation size correlated positively with temperature. There was a clear trend towards the correlation of time over 44°C and ablation depth. Maximum temperatures did not significantly correlate with coagulation size. Laser ablation of lung tumors using the IHLP (isolated human lung perfusion) model represents a possible method for evaluating ex vivo the interrelationships of temperature, time of temperature exposure, and resulting coagulation. [ABSTRACT FROM AUTHOR]

Purpose: To assess the reliability of trypan blue (TB) and calcein AM/ethidium homodimer-1 (CaAM/EthD-1) staining to evaluate the viability of fresh and thawed human ovarian follicles. Methods: Isolated follicles from fresh and thawed cortex were stained using TB versus CaAM/EthD-1 methods ( n = 10 patients). Measurements were performed by two independent observers. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements were tested by the Wilcoxon test. Results: Inter-observer reliability was excellent for each method. Nevertheless, it was even better with the TB method (ICC = 0.83) compared with CaAM/EthD-1 (ICC = 0.75). Moreover, the ICCs for viability measurements using the two methods were good for each observer (observer 1: ICC = 0.49; observer 2: ICC = 0.40). Conclusion: Compared with CaAM/EthD-1, TB appears to be more reliable as a staining method for follicle viability evaluation. TB staining is a quick and useful method, complementary to histological analysis for quality control in ovarian tissue cryopreservation. [ABSTRACT FROM AUTHOR]

Abstract: Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis. [Copyright &y& Elsevier]

Pulsed electric fields (PEF) nonthermally induce cell membrane permeabilization and thereby improve dehydration and extraction efficiencies in food plant materials. Effects of electrical field strength and number of pulses on plant tissue integrity have been studied extensively. Two previous studies on the effect of pulse frequency, however, did not provide a clear view: one study suggested no effect of frequency, while the other found a greater impact on tissue integrity at lower frequency. This study establishes the effect of pulse frequency on integrity of onion tissues. Changes in electrical characteristics, ion leakage, texture parameters, and percent weight loss were quantified for a wide range of pulse frequencies under conditions of fixed field strength and pulse number. Optical microscopy and viable-cell staining provided direct visualization of effects on individual cells. The key finding is that lower frequencies ( f < 1 Hz) cause more damage to tissue integrity than higher frequencies ( f= 1 to 5000 Hz). Intriguingly, the optical microscopy observations demonstrate that the speed of intracellular convective motion (that is, cytoplasmic streaming) following PEF application is strongly correlated with PEF frequency. We provide the first in situ visualization of the intracellular consequence of PEF at different frequencies in a plant tissue. We hypothesize that cytoplasmic streaming plays a significant role in moving conductive ionic species from permeabilized cells to the intercellular space between plant cells, making subsequent pulses more efficacious at sufficiently low frequencies. The results suggest that decreasing the pulse frequency in PEF may minimize the number of pulses needed to achieve a desired amount of permeabilization, thus lowering the total energy consumption. PEF cause pores to be formed in plant cell membranes, thereby improve moisture removal and potential extraction of desirable components. This study used in situ microscopic evaluation of onion cells, as they were pulsed with electric fields at different frequencies, to determine whether frequency was an important parameter. We illustrate that membranes were more effectively broken at lower frequencies as compared to higher frequencies. Application of this information will allow for improved design of PEF systems for more energy efficient dehydration or extraction of plant tissues. [ABSTRACT FROM AUTHOR]

1. Many internally-fertilizing animals produce seminal fluid which is transferred along with sperm during mating. Seminal fluid typically contains a diverse range of chemicals that coordinate sperm storage, moderate sperm motility, provide advantages in sexual selection and influence female physiology. 2. Seminal fluid is well-studied in Drosophila melanogaster, a species in which it has been suggested to ‘incapacitate’ the sperm of rival males (e.g. by killing them) and thereby provide an advantage in sperm competition. This hypothesis has been tested several times over many years, but different studies have yielded conflicting conclusions. Here, I use fluorescent staining to directly measure the effects of D. melanogaster seminal fluid on the survival of sperm from the same male or from a rival. The results suggest that seminal fluid improves sperm survival, even if the sperm are from a different male. This study therefore provides strong evidence that seminal fluid does not kill rival sperm, and instead can actually protect them. This study also tested whether chemicals in the female reproductive tract harm sperm as in another Drosophila species, but found no evidence of this. 3. These findings suggest that residual seminal fluid inside females could benefit the sperm of subsequent mates, affecting the outcome of sperm competition and influencing the evolution of ejaculates and mating systems. [ABSTRACT FROM AUTHOR]

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 μg/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 μg/ml or greater (ANOVA, Fisher's PLSD test), and complete killing of the cells at 250 μg/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. [ABSTRACT FROM AUTHOR]

Abstract: The effectiveness of mycorrhizal colonisation of four fungal isolates of different origin was tested in roots of Festuca rubra L. and Plantago lanceolata L. cultivated on three zinc waste substrata of different toxicity. F. rubra showed a much better survival rate than P. lanceolata. The effectiveness of mycorrhizal colonisation varied between the fungal isolates introduced. The highest values of the mycorrhizal parameters were found in roots inoculated with the Glomus claroideum strain originating from the industrial waste. Arbuscular richness, assessed in roots stained for viable fungal structures, was demonstrated to be the most sensitive parameter, showing statistically important differences between plants, artificially inoculated and those colonised only by the indigenous mycorrhizal fungi. This parameter was also useful to compare the substrata concerning their toxicity, and the results of the viability tests correlated well with the “availability” of the heavy metals. [Copyright &y& Elsevier]

Abstract: Microscopic techniques ranging from epifluorescence microscopy to confocal laser scanning microscopy (CLSM) and two photon excitation laser scanning microscopy (TPE-LSM) combined with fluorescent stains can help to evaluate complex microbial aggregates such as activated sludge flocs. To determine the application limits of these microscopic techniques, activated sludge samples from three different sources were evaluated after staining with a fluorescent viability indicator (Baclight Bacterial Viability Kit, Molecular Probes). Image analysis routines were developed to quantify overall amounts of red and green stained cells, location of stained cells within the flocs, and the spatial organization in clusters and filaments. It was found that the selection of the appropriate microscopic technique depends strongly on the type of microbial aggregates being analyzed. For flocs with high cell density, the use of TPE-LSM is preferred, since it provides a clearer image of the internal structure of the aggregate. Epifluorescence microscopy did not allow to reliably quantify red stained cells in dense aggregates. CLSM did not adequately image the internal filamentous structure and the location of stained cells within dense flocs. However, for typical activated sludge flocs epifluorescence and CLSM proved adequate. [Copyright &y& Elsevier]

Abstract: Pseudomonas fluorescens SBW25, a plant growth promoting bacterium, has been widely studied due to its potential as an inoculum for improving crop yields. Environmental inoculants are usually applied on seeds or directly to soil and to effectively promote plant growth they need to be viable and active. However, it is difficult to study the physiological status of specific microorganisms in complex environments, such as soil. In this study, our aim was to use molecular tools to specifically monitor the physiological status of P. fluorescens SBW25 in soil and in pure cultures incubated under different nutritional conditions. The cells were previously tagged with marker genes (encoding green fluorescent protein and bacterial luciferase) to specifically track the cells in environmental samples. The physiological status of the cells was determined using the viability stains 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and propidium iodide (PI), which stain active and dead cells, respectively. Luciferase activity was used to monitor the metabolic activity of the population. Most of the cells died after incubation for nine days in nutrient rich medium. By contrast when incubated under starvation conditions, most of the population was not stained with CTC or PI (i.e. intact but inactive cells), indicating that most of the cells were presumably dormant. In soil, a large fraction of the SBW25 cell population became inactive and died, as determined by a decline in luciferase activity and CTC-stained cells, an increase in PI-stained cells, and an inability of the cells to be cultured on agar medium. However, approximately 60% of the population was unstained, presumably indicating that the cells entered a state of dormancy in soil similar to that observed under starvation conditions in pure cultures. These results demonstrate the applicability of this approach for monitoring the physiological status of specific cells under stress conditions, such as those experienced by environmental inoculants in soil. [Copyright &y& Elsevier]

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean ( Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured cells was greater ( P<0.001) with filtrates prepared from F. solani f. sp. glycines than from other F. solani isolates, and dead cells increased over time and with higher concentrations of culture filtrate. Cuttings of soybean seedlings with their stems immersed in culture filtrates of F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when immersed in filtrates of other isolates. There was a positive correlation ( r=0.94, P<0.001) between soybean foliar symptom severity and percentage of stained soybean suspension-cultured cells. Both methods were used to determine the phytotoxicity of fungal culture filtrates. [ABSTRACT FROM AUTHOR]