Search

Abstract β-TCP ceramics are versatile bone substitute materials and show many interactions with cells of the monocyte-macrophage-lineage. The possibility of monocytes entering microporous β-TCP ceramics has however not yet been researched. In this study, we used a model approach to investigate whether monocytes might enter β-TCP, providing a possible explanation for the origin of CD68-positive osteoclast-like giant cells found in earlier works. We used flow chambers to unidirectionally load BC, PRP, or PPP into slice models of either 2 mm or 6 mm β-TCP. Immunofluorescence for CD68 and live/dead staining was performed after the loading process. Our results show that monocytes were present in a relevant number of PRP and BC slices representing the inside of our 2 mm slice model and also present on the actual inside of our 6 mm model. For PPP, monocytes were not found beyond the surface in either model. Our results indicate the possibility of a new and so far neglected constituent in β-TCP degradation, perhaps causing the process of ceramic degradation also starting from inside the ceramics as opposed to the current understanding. We also demonstrated flow chambers as a possible new in vitro model for interactions between blood and β-TCP.

Abstract Fluorescence analysis of β-TCP ceramics is often used to describe cells found on said ceramics. However, we found, to our knowledge, so far undescribed artifacts which might sometimes be hard to differentiate from cells due to shape and fluorescence behavior. We tried prolonged ultrasound washing as well as Technovit 9100 fixation to reduce these artifacts. While untreated dowels showed no reduction in artifacts no matter the further treatment, Technovit fixation reduced the artifacts with even further reduction achieved by mechanical cleaning. As a consequence, scientists working with these dowels and likely even other types should try to avoid creating false positive results by considering the existence of these artifacts, checking additional filters for unusual fluorescence and by reducing them by using Technovit fixation when possible.

β-TCP ceramics are versatile bone substitute materials and show many interactions with cells of the monocyte-macrophage-lineage. The possibility of monocytes entering microporous β-TCP ceramics has however not yet been researched. In this study, we used a model approach to investigate whether monocytes might enter β-TCP, providing a possible explanation for the origin of CD68-positive osteoclast-like giant cells found in earlier works. We used flow chambers to unidirectionally load BC, PRP, or PPP into slice models of either 2 mm or 6 mm β-TCP. Immunofluorescence for CD68 and live/dead staining was performed after the loading process. Our results show that monocytes were present in a relevant number of PRP and BC slices representing the inside of our 2 mm slice model and also present on the actual inside of our 6 mm model. For PPP, monocytes were not found beyond the surface in either model. Our results indicate the possibility of a new and so far neglected constituent in β-TCP degradation, perhaps causing the process of ceramic degradation also starting from inside the ceramics as opposed to the current understanding. We also demonstrated flow chambers as a possible new in vitro model for interactions between blood and β-TCP. [ABSTRACT FROM AUTHOR]

Fluorescence analysis of β-TCP ceramics is often used to describe cells found on said ceramics. However, we found, to our knowledge, so far undescribed artifacts which might sometimes be hard to differentiate from cells due to shape and fluorescence behavior. We tried prolonged ultrasound washing as well as Technovit 9100 fixation to reduce these artifacts. While untreated dowels showed no reduction in artifacts no matter the further treatment, Technovit fixation reduced the artifacts with even further reduction achieved by mechanical cleaning. As a consequence, scientists working with these dowels and likely even other types should try to avoid creating false positive results by considering the existence of these artifacts, checking additional filters for unusual fluorescence and by reducing them by using Technovit fixation when possible. [ABSTRACT FROM AUTHOR]

The study was carried out on four non-tidal sandy marine beaches located on the Polish part of the southern Baltic Sea coast. We applied a LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Invitrogen™) method to determine the abundance of live and dead bacteriopsammon. Live psammon bacteria cells constituted 31–53% of the total number of bacteria inhabiting sand of the studied beaches. Abundance of live and dead psammon bacteria generally differed along the horizontal profile in all beaches. The maximum density of bacteria was noted in the dune and the middle part of the beach (dry zones) and the minimum in wet zones, i.e., under seawater surface and at the swash zone. Generally along the vertical profile, the highest numbers of two studied bacterial groups were noted in the surface sand layer, while with increasing sediment depth their numbers significantly decreased. The abundance of live and dead bacteria showed a distinct seasonal variation. [ABSTRACT FROM AUTHOR]

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

Electrocautery is a common surgical technique and is often used during shoulder arthroplasty to elevate or transect the subscapularis tendon. The relative amount of tissue damage caused by cautery as opposed to sharp transection is not currently known. The purpose of this study was to examine local tissue damage resulting from electrocautery vs. sharp transection with a scalpel. We hypothesized that the electrosurgical unit would cause higher collateral tissue damage and cell death compared with sharp transection. Twelve cadaveric ovine shoulders were randomized to either the electrosurgical or sharp transection group. The infraspinatus tendon was isolated, and a partial-thickness transection was made using either a monopolar electrosurgical device (Bovie) or No. 10 scalpel blade. Tendon explants were then visualized with confocal microscopy to evaluate tissue architecture. A live/dead assay was performed using microscopy imaging analysis software. Comparisons between Bovie and scalpel transection were made using the Mann-Whitney U test, and the cell death percentage at standardized distances from the transection site was compared between groups using a mixed-model analysis. Significance was defined at P <.05. The cellular and tendon fibril architecture was well maintained beyond the scalpel transection site, whereas Bovie transection disrupted the architecture beyond its transection path. The percentage of dead cells in the Bovie group (74.9% ± 31.2%) was significantly higher than that in the scalpel group (27.6% ± 29.9%, P =.0004). Compared with the transection site, the cell death percentage after Bovie transection significantly declined at 2.5 mm whereas that after scalpel transection significantly declined at 1 mm from the transection site. There was a significantly higher dead cell percentage in the Bovie transection group, indicating extensive damage beyond the local incision site, compared with sharp transection. Electrosurgical transection of the ovine infraspinatus tendon ex vivo caused higher cell death and greater tissue architecture disruption compared with sharp scalpel transection. [ABSTRACT FROM AUTHOR]

Candida auris is a potential multidrug-resistant pathogen able to cause biofilm-associated outbreaks, where frequently indwelling devices are the source of infections. The number of effective therapies is limited; thus, new, even-combination-based strategies are needed. Therefore, the in vitro efficacy of caspofungin with posaconazole against FKS wild-type and mutant Candida auris isolates was determined. The interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss model, and a LIVE/DEAD assay. Planktonic minimum inhibitory concentrations (pMICs) for the caspofungin–posaconazole combination showed a 4- to 256-fold and a 2- to 512-fold decrease compared to caspofungin and posaconazole alone, respectively. Sessile minimum inhibitory concentrations (sMICs) for caspofungin and posaconazole in combination showed an 8- to 128-fold and a 4- to 512-fold decrease, respectively. The combination showed synergy, especially against biofilms (FICIs were 0.033–0.375 and 0.091–0.5, and Bliss cumulative synergy volumes were 6.96 and 32.39 for echinocandin-susceptible and -resistant isolates, respectively). The caspofungin-exposed (4 mg/L) C. auris biofilms exhibited increased cell death in the presence of posaconazole (0.03 mg/L) compared to untreated, caspofungin-exposed and posaconazole-treated biofilms. Despite the favorable effect of caspofungin with posaconazole, in vivo studies are needed to confirm the therapeutic potential of this combination in C. auris-associated infections.

Background: The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities. Results: We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 105 CFU and that Benzonase digest is not sufficient to overcome this bias. Conclusions: The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. BtfPryzAHp2_2685tzMGyk Video abstract [ABSTRACT FROM AUTHOR]

Abstract While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

The paper compares two methods of distinguishing between alive and dead cells by differentiation on the basis of their membrane structure: LIVE/DEAD flow cytometry and PMA-qPCR. LIVE/DEAD flow cytometry was established using the LIVE/DEAD(®) BacLight™ Bacterial Viability Kit with different ratios of Legionella pneumophila and Escherichia coli cells with intact and compromised membranes (heat treated). The PMA-qPCR method was tested and modified, and results were compared with those from LIVE/DEAD flow cytometry using L. pneumophila cells. Ratios of membrane intact to membrane compromised cells were well shown by LIVE/DEAD flow cytometry in all combinations. PMA-qPCR seems to work best in even mixed ratios (1:1) of intact and compromised cells. In other respects, we noticed an overestimation of intact cells in the samples which contained a high percentage of membrane compromised cells, and an underestimation of intact cells in samples with a small percentage of membrane compromised cells. However, looking at total counts instead of ratios, the results were within an order of magnitude. This implies that the use of PMA-qPCR is appropriate only for a qualitative analysis to monitor the success of a process such as disinfection. Furthermore, we were able to assess that both methods have advantages and disadvantages: LIVE/DEAD flow cytometry as applied in this study works well on some bacteria monocultures, but does not distinguish between bacteria species. The PMA-qPCR method allows the possibility of distinguishing between membrane intact cells and membrane compromised cells and can be used to screen for specific bacteria. [ABSTRACT FROM AUTHOR]

Abstract: A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles. This study compared the antimicrobial efficacy of this novel dressing to two commercially available silver dressings; Acticoat™ and PolyMem Silver©. Three different antimicrobial tests were used: disc diffusion, broth culture, and the Live/Dead© Baclight™ bacterial viability assay. Burn wound pathogens (P. aeruginosa, MSSA, A. baumannii and C. albicans) and antibiotic resistant strains (MRSA and VRE) were tested. All three antimicrobial tests indicated that Acticoat™ was the most effective antimicrobial agent, with inhibition zone lengths of 13.9–18.4mm. It reduced the microbial inocula below the limit of detection (102 CFU/ml) and reduced viability by 99% within 4h. PolyMem Silver© had no zone of inhibition for most tested micro-organisms, and it also showed poor antimicrobial activity in the broth culture and Live/Dead© Baclight™ assays. Alarmingly, it appeared to promote the growth of VRE. The silver hydrogel reduced most of the tested microbial inocula below the detection limit and decreased bacterial viability by 94–99% after 24h exposure. These results support the possibility of using this novel silver hydrogel as a burn wound dressing in the future. [ABSTRACT FROM AUTHOR]

Candida auris is a potential multidrug-resistant pathogen able to cause biofilm-associated outbreaks, where frequently indwelling devices are the source of infections. The number of effective therapies is limited; thus, new, even-combination-based strategies are needed. Therefore, the in vitro efficacy of caspofungin with posaconazole against FKS wild-type and mutant Candida auris isolates was determined. The interactions were assessed utilizing the fractional inhibitory concentration indices (FICIs), the Bliss model, and a LIVE/DEAD assay. Planktonic minimum inhibitory concentrations (pMICs) for the caspofungin-posaconazole combination showed a 4- to 256-fold and a 2- to 512-fold decrease compared to caspofungin and posaconazole alone, respectively. Sessile minimum inhibitory concentrations (sMICs) for caspofungin and posaconazole in combination showed an 8- to 128-fold and a 4- to 512-fold decrease, respectively. The combination showed synergy, especially against biofilms (FICIs were 0.033-0.375 and 0.091-0.5, and Bliss cumulative synergy volumes were 6.96 and 32.39 for echinocandin-susceptible and -resistant isolates, respectively). The caspofungin-exposed (4 mg/L) C. auris biofilms exhibited increased cell death in the presence of posaconazole (0.03 mg/L) compared to untreated, caspofungin-exposed and posaconazole-treated biofilms. Despite the favorable effect of caspofungin with posaconazole, in vivo studies are needed to confirm the therapeutic potential of this combination in C. auris -associated infections.

Two essential requirements for probiotic bifidobacteria are that they be 'live' and have 'colonization' ability, following FAO/WHO guideline recommendations. The amount of research on the adhesion ability of bifidobacteria compares poorly with that of other probiotic bacteria, such as lactobacilli. The aim of the present study was to determine how gastrointestinal conditions affect the adhesion ability of bifidobacteria, and to investigate the relationship between the adhesion ability and the live/dead state of bifidobacteria. The adhesion ability of Bifidobacterium animalis KLDS2.0603 that had been subjected to the digestive enzymes, pepsin, trypsin, and proteinase K, was decreased significantly, but these treatments did not significantly change the strain's survival rates, which were 98.78%, 97.60%, and 97.63% respectively. B. animalis KLDS2.0603 subjected to LiCl retained its adhesion ability but had a lower survival rate (59.28%) than the control group ( P<0.01). B. animalis KLDS 2.0603 subjected to sodium metaperiodate exhibited higher adhesion ability than the control group ( P<0.01), but the bacterial cells were killed totally. The results of transmission electron microscopy and laser scanning confocal microscopy showed that live/dead state of bifidobacteria was not one of the main factors that affected the adhesion ability of bifidobacteira, and that the substances affecting the adhesion ability of bifidobacteria were on the outer surface layer of the bifidobacterial cells. Our results also indicated that the substances related to the adhesion ability of bifidobacteria are proteinaceous. The above results will help us to understand the adhesion and colonization processes of bifidobacteria in the human gastrointestinal tract. [ABSTRACT FROM AUTHOR]

biofilms with different proportions of viable but nonculturable bacteria were used to show that SYBR green (SYBR) may be used as a probe to evaluate the bacterial physiological state using flow cytometry. Biofilms grown in excess glucose presented significantly higher proportions of dormant bacteria than biofilms grown in excess glucose with buffered pH conditions or with exponential-phase planktonic cultures. Bacteria obtained from biofilms with high or low proportions of viable but nonculturable cells were further cultured in broth medium and stained with SYBR at different time points. An association between bacterial growth and SYBR staining intensity was observed. In addition, bacteria presenting higher SYBR fluorescence intensity also stained more intensely with cyanoditolyl tetrazolium chloride, used as a probe to evaluate cellular metabolism. Accordingly, planktonic bacteria treated with rifampicin, an inhibitor of bacterial RNA transcription, presented lower SYBR and cyanoditolyl tetrazolium chloride staining intensity than nontreated bacteria. Overall, our results indicate that SYBR, in addition to being used as a component of LIVE/DEAD stain, may also be used as a probe to evaluate the physiological state of S. epidermidis cells. [ABSTRACT FROM AUTHOR]

Abstract: Recent developments in host–microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0–4.0log/mL, whereas a 5.0–5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria. [Copyright &y& Elsevier]

The permanent ice covers of the McMurdo Dry Valley lakes, Antarctica, are colonized by a diverse microbial assemblage. We collected ice cores from Lakes Fryxell, Hoare and Bonney. Propidium monoazide (PMA) was used in combination with quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) to examine membrane integrity of prokaryotes in these extreme environments. PMA selectively penetrates cells with compromised membranes and modifies their DNA resulting in the suppression of PCR amplification. Our results based on analysis of 16S rRNA genes demonstrate that despite the hostile conditions of the Dry Valleys, the permanent ice covers of the lakes support a 'potentially viable' microbial community. The level of membrane integrity, as well as diversity, was higher in samples where sediment was entrapped in the ice cover. Pronounced differences in the fraction of cells with intact and compromised cell membranes were found for Lake Fryxell and east lobe of Lake Bonney, both expressed in differences in DGGE banding patterns and qPCR signal reductions. Limitations in the ability to distinguish between intact or compromised cells occurred in samples from Lake Hoare and west lobe of Lake Bonney due to low DNA template concentrations recovered from the samples. [ABSTRACT FROM AUTHOR]

Abstract: Single-walled carbon nanotubes (SWCNT), fullerenes (C60), carbon black (CB), nC60, and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C60, nC60, and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96® AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue® (CTB), CytoTox One™ (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R2 value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment. [Copyright &y& Elsevier]

The increase in the number of revision surgeries after a total knee replacement surgery reaches 19%. One of the reasons for the majority of revisions relates to the debris of the ultra-high molecular weight polyethylene that serves to facilitate the sliding between the femoral and tibial components. This paper addresses the biomechanical properties of ULTEMTM 1010 in a totally new knee replacement design, based on one of the commercial models of the Stryker manufacturer. It is designed and produced through additive manufacturing that replaces the tibial component and the polyethylene in such a way as to reduce the pieces that are part of the prosthetic assembly to only two: the femoral and the tibial (the so-called "two-component knee prosthesis"). The cytotoxicity as well as the live/dead tests carried out on a series of biomaterials guarantee the best osteointegration of the studied material. The finite element simulation method guarantees the stability of the material before a load of 2000 N is applied in the bending angles 0°, 30°, 60°, 90°, and 120°. Thus, the non-metallic prosthetic material and approach represent a promising alternative for metal-allergic patients. [ABSTRACT FROM AUTHOR]

Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.

Ozone was applied to return activated sludge in full-scale to study how ozone impacts filamentous bacteria viability (Live/Dead ® ). Additionally, the ozonated sludges were subjected to anaerobic digestion trials and analysis of micropollutants (MPs). Ozone treatment (3-4.8 g O 3 /kg total suspended solids) improved the settling properties of the sludge by lowering the diluted sludge volume with 7-35%. Ozone inactivated filamentous bacteria outside the floc structures and the fraction of inactivated filaments increased with an increasing ozone dose. It was observed that ozone treatment may act selectively towards different types of filaments. With respect to the two dominating morphotypes present, Type 0041 filamentous bacteria were found to be more resistant to ozone attack than Microthrix parvicella . Thus, higher ozone doses may be required to mitigate sludge bulking caused by Type 0041 filaments. No effects could be discerned by ozone addition on neither the methane production of the sludge nor on the concentrations of MPs analysed for this study. The lack of effect on both methane production and micropollutant removal was deemed to be caused by insufficient ozone doses.

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles. This study compared the antimicrobial efficacy of this novel dressing to two commercially available silver dressings; Acticoat™ and PolyMem Silver(®). Three different antimicrobial tests were used: disc diffusion, broth culture, and the Live/Dead(®) Baclight™ bacterial viability assay. Burn wound pathogens (P. aeruginosa, MSSA, A. baumannii and C. albicans) and antibiotic resistant strains (MRSA and VRE) were tested. All three antimicrobial tests indicated that Acticoat™ was the most effective antimicrobial agent, with inhibition zone lengths of 13.9-18.4mm. It reduced the microbial inocula below the limit of detection (10(2)CFU/ml) and reduced viability by 99% within 4h. PolyMem Silver(®) had no zone of inhibition for most tested micro-organisms, and it also showed poor antimicrobial activity in the broth culture and Live/Dead(®) Baclight™ assays. Alarmingly, it appeared to promote the growth of VRE. The silver hydrogel reduced most of the tested microbial inocula below the detection limit and decreased bacterial viability by 94-99% after 24h exposure. These results support the possibility of using this novel silver hydrogel as a burn wound dressing in the future., (Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.)