Microscopic Cell Counts Research Articles

The effect of pH on the acute toxicity of phenanthrene in a marine microalgae Chlorella salina

Phenanthrene is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) found in continental shelf environment of China and is on the EPA’s Priority Pollutant list. In this study, the effects of phenanthrene on marine algal growth rate were determined after 96-h exposure at pH 6.0, 7.0, 8.0, 9.0, and 10.0 in seawater of salinity 35. Two measuring techniques to assess growth inhibition were also compared using prompt fluorescence and microscopic cell count. The results showed that the toxicity of phenanthrene increased significantly (p < 0.05) with decreasing pH, with the nominal concentration required to inhibit growth rate by 50%, EC50, decreasing from 1.893 to 0.237 mg L−1 as pH decreased from 9.0 to 6.0, with a decrease higher than 55% from 10.0 to 9.0. In addition, the nominal EC50 values calculated in this study were at the same range of some environmental concentrations of phenanthrene close to areas of crude oil exploration. Based on the two measuring techniques, the results showed that cell count and fluorescence measurement were significantly different (p < 0.05), and the nominal EC50 values calculated with cell count measurement were significantly higher than fluorescence measurement at pH 8.0, 9.0 and 10.0. In conclusion, the present studies confirmed that acidification of seawater could affect the toxicity of phenanthrene to this species of microalgae, and which encouraged further studies involving responses of marine organisms to ocean acidification.

Milk cellularity and intramammary infections in primiparous and multiparous Lacaune ewes during early lactation

Despite the vast literature on postpartum mastitis in cattle, there is a demand for and lack of studies on the cellularity dynamics and cellular populations in milk and the occurrence of mastitis in ewes during the postpartum period. Thus, the aim of the present study was to evaluate the dynamics of cellularity in milk/colostrum and of mastitis in primiparous and multiparous Lacaune ewes during early lactation. For this purpose, 492 milk/colostrum samples were collected from the udder halves of 20 primiparous ewes and 21 multiparous ewes at the following six time points: day of parturition (T1) and one day (T2), three days (T3), seven days (T4), 15 days (T5), and 30 days (T6) after parturition. The milk and colostrum samples were subjected to microbiological examination, microscopic and automatic somatic cell counts (SCCs), differential somatic cell counts, and the California Mastitis test (CMT). The multiparous ewes had a higher prevalence of intramammary infections than the primiparous ewes during the postpartum period. Non-aureus staphylococci bacteria were the main mastitis etiological agents isolated in the present study. Furthermore, one of the most important findings of our study was that 35% of the udder halves of the primiparous ewes were infected at parturition, suggesting potential concerns for mastitis control programs. Finally, the SCC did not appear to be a good indicator of inflammation during early lactation because this measure was influenced by the parity and the sampling period. Moreover, the predictive values of the CMT and neutrophil counts were slightly better than those of the SCC during the immediate postpartum period.

Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa.

A variety of direct and indirect methods have been used to quantify planktonic and biofilm bacterial cells. Direct counting methods to determine the total number of cells include plate counts, microscopic cell counts, Coulter cell counting, flow cytometry, and fluorescence microscopy. However, indirect methods are often used to supplement direct cell counting, as they are often more convenient, less time-consuming, and require less material, while providing a number that can be related to the direct cell count. Herein, an indirect method is presented that uses fluorescence emission intensity as a proxy marker for studying bacterial accumulation. A clinical strain of Pseudomonas aeruginosa was genetically modified to express a green fluorescent protein (PA14/EGFP). The fluorescence intensity of EGFP in live cells was used as an indirect measure of live cell density, and was compared with the traditional cell counting methods of optical density (OD600) and plate counting (colony-forming units (CFUs)). While both OD600 and CFUs are well-established methods, the use of fluorescence spectroscopy to quantify bacteria is less common. This study demonstrates that EGFP intensity is a convenient reporter for bacterial quantification. In addition, we demonstrate the potential for fluorescence spectroscopy to be used to measure the quantity of PA14/EGFP biofilms, which have important human health implications due to their antimicrobial resistance. Therefore, fluorescence spectroscopy could serve as an alternative or complementary quick assay to quantify bacteria in planktonic cultures and biofilms.

Development of a qPCR assay to detect and quantify ichthyotoxic flagellates along the Norwegian coast, and the first Norwegian record of Fibrocapsa japonica (Raphidophyceae)

Blooms of ichthyotoxic microalgae pose a great challenge to the aquaculture industry world-wide, and there is a need for fast and specific methods for their detection and quantification in monitoring programs. In this study, quantitative real-time PCR (qPCR) assays for the detection and enumeration of three ichthyotoxic flagellates: the dinoflagellate Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup and the two raphidophytes Heterosigma akashiwo (Hada) Hada ex Hara & Chihara and Fibrocapsa japonica Toriumi & Takano were developed. Further, a previously published qPCR assay for the dinoflagellate Karlodinium veneficum (Ballantine) Larsen was used. Monthly samples collected for three years (Aug 2009–Jun 2012) in outer Oslofjorden, Norway were analysed, and the results compared with light microscopy cell counts. The results indicate a higher sensitivity and a lower detection limit (down to 1 cell L−1) for both qPCR assays. Qualitative and semi-quantitative results were further compared with those obtained by environmental 454 high throughput sequencing (HTS, metabarcoding) and scanning electron microscopy (SEM) examination from the same samplings. All four species were detected by qPCR and HTS and/or SEM in outer Oslofjorden (Aug 2009–Jun 2012); Karlodinium veneficum was present year-round, whereas Karenia mikimotoi, Heterosigma akashiwo and Fibrocapsa japonica appeared mainly during the autumn in all three years. This is the first observation of Fibrocapsa japonica in Norwegian coastal waters. This species has previously been recorded off the Swedish west coast and German Bight, which may suggest a northward dispersal.

Critical comparison of molecular methods for detection and enumeration of the harmful algal species, Heterosigma akashiwo, in environmental water samples

Molecular methods such as quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) enable a more rapid and specific enumeration of harmful algal species compared to microscopic cell counts. Integrating these methods into routine monitoring and management strategies, however, has been slow. While comparisons to microscopy have been made, direct comparisons between molecular methods using environmental samples are sparse. Here, we directly compare qPCR to SHA for enumerating the harmful algal species, Heterosigma akashiwo, in environmental samples collected in Delaware’s inland bays. To ensure comparability, a single cellular homogenate was generated from field samples and split for analysis by qPCR and SHA. Results show a significant correlation between qPCR and SHA when Heterosigma is above bloom levels (1 × 105 cells L−1), but not during non-bloom conditions. qPCR and SHA were also more highly correlated when samples were collected at lower temperatures (< 25 °C) and/or with high levels of chlorophyll a (greater than or equal to 30 μg L−1), independent of Heterosigma cell concentration. There was no evidence of cross-reactivity in primers and probes for H. akashiwo during blooms of the closely related species, Chattonella subsalsa. However, qPCR to SHA ratios were elevated during blooms of other phytoplankton species, suggesting suppression of the SHA signal or enhancement of qPCR. Results of this study may have significant implications for research, where precise evaluations of cell numbers are often required. However, precise cell counts at non-bloom levels may not be as critical to management, suggesting either technique could be incorporated into rapid and effective decision making.

Diagnosing mastitis in early lactation: use of Somaticell®, California mastitis test and somatic cell count

The objective of the present study was to evaluate different methods for indirectly diagnosing mastitis during the postpartum period. These methods were: automatic and microscopic somatic cell counting (SCC), the California Mastitis Test (CMT) and Somaticell®. A total of 538 milk samples from 34 cows were used. These were collected at six times: day of parturition (M1) and 3 (M2), 7 (M3), 15 (M4), 21 (M5) and 30 (M6) days after parturition. Automatic and microscopic SCC, CMT and Somaticell® were all able to detect mastitis during the immediate postpartum period (up to 3 days postpartum). However, higher cut-off values should be applied to automatic and microscopic SCC. The negative score (score 0) of CMT was considered to be the best cut-off point at all times. Moreover, the values found using the Somaticell® test should not be used to presume the automatic SCC values, since there are discrepancies between the values of Somaticell® and automatic and microscopic SCC. It can be concluded that the different methods evaluated here to milk cellularity can be applied for diagnosing bovine mastitis, even during the immediate postpartum period, when there is greater cellularity, such as in the colostrum and transition milk.

Molecular detection of harmful cyanobacteria and expression of their toxin genes in Dutch lakes using multi-probe RNA chips

Harmful cyanobacterial blooms are a major threat to water quality and human health. Adequate risk assessment is thus required, which relies strongly on comprehensive monitoring. Here, we tested novel multi-probe RNA chips developed in the European project, μAqua, to determine the abundance of harmful cyanobacterial species and expression of selected toxin genes in six Dutch lakes. All of the targeted cyanobacterial genera, except for Planktothrix, were detected using the microarray, with predominance of Dolichospermum and Microcystis signals, of which the former was found across all sites and detected by the probes for Anabaena where it was formerly placed. These were confirmed by microscopic cell counts at three sites, whereas at the other sites, microscopic cell counts were lower. Probe signals of Microcystis showed larger variation across sites but also matched microscopic counts for three sites. At the other sites, microscopic counts were distinctly higher. We detected anatoxin-a in the water at all sites, but unfortunately no genes for this toxin were on this generation of the toxin array. For microcystins, we found none or low concentrations in the water, despite high population densities of putative microcystin producers (i.e. Microcystis, Dolichospermum). The described method requires further testing with a wider range of cyanobacterial communities and toxin concentrations before implementation into routine cyanobacterial risk assessment. Yet, our results demonstrate a great potential for applying multi-probe RNA chips for species as well as toxins to eutrophic waters with high cyanobacterial densities as a routine monitoring tool and as a predictive tool for toxin potential.

Asymptomatic bacteriuria in pregnant women attending Boo-Ali Hospital Tehran Iran: Urine analysis vs. urine culture.

BackgroundAsymptomatic bacteriuria is one of the common problems in pregnancy. Asymptomatic bacteriuria is associated with pyelonephritis, preterm labor and low birth weight infants. The physiological and anatomical changes in pregnancy facilitate urinary tract infection (UTI) during pregnancy. Several tests are available for diagnosis of asymptomatic bacteriuria. The urine culture is a gold standard diagnostic test for asymptomatic bacteriuria but it is expensive and time-consuming. Screening methods may be useful in detecting high-risk pregnant women for asymptomatic bacteriuria.ObjectiveThe aim of the present study was to compare urine analysis as a rapid screening test to urine culture in diagnosis of asymptomatic bacteriuria.MethodsA total of 123 pregnant women attending the obstetrics clinic of Boo-Ali hospital in Tehran, Iran from March 2013 to September 2014 were included in the present diagnostic cross-sectional study. One hundred twenty three mid-stream urine samples were inoculated into cultures and were processed by dipstick (nitrite test and leucocyte esterase test) and microscopic pus cell count. The sensitivity, specificity, positive predictive value and negative predictive value of nitrite test, leucocyte esterase test and microscopic pus cell count were compared with urine culture in diagnosis of asymptomatic bacteriuria by using SPSS version 19.ResultsOf 123 urine samples, significant asymptomatic bacteriuria (≥104 cfu/Ml) was detected in 8 (6.5%) subjects. The sensitivity and specificity of nitrite test were 37% and 100% respectively. The sensitivity of pus cell count alone and leucocyte esterase test alone were 100% but the specificity of them were 64% and 65% respectively. We found high negative predictive value by Pus cell count and the leucocyte esterase test (100%) and low positive predictive value by them (16% and 17% respectively).ConclusionUrine culture is the most useful test for diagnosis of asymptomatic bacteriuria. None of our screening tests had a sensitivity and specificity of 100%, whereas we can only refer the pregnant women with positive leucocyte esterase test and significant pyuria to the urine culture.

Interactions of nanoscale zero valent iron and iron reducing bacteria in remediation of trichloroethene

Absract In this work we investigated the interactions between the nanoscale zero valent iron (nZVI) and iron reducing bacteria (IRB) during the remediation of trichloroethene (TCE). Dehalogenation of TCE was examined using nZVI NANOFER 25 (N25) and the Gram-negative bacterium Shewanella algae CCM 4595 in liquid and soil slurry microcosms. The rates of dehalogenation were determined by gas chromatography. The interactions between N25 and S. algae were investigated using X-Ray diffraction (XRD), ferrozine test, Tandem Scanning Confocal Microscope (TSCM) and viable cell counts. Results showed that S. algae impaired TCE removal by N25. TCE dehalogenation rates in liquid microcosms were 17 times faster in absence than in presence of S. algae, and 8 times faster in soil slurry microcosms. The concentrations of dissolved Fe2+ and Fe3+ ions in the N25 + S. algae microcosms were an order of magnitude higher than that in bacteria-free microcosms. Moreover, the XRD data showed that the presence of S. algae significantly increases the depletion of reactive Fe0 and causes the formation of poorly soluble and insoluble Fe3+ oxide-hydroxides that can inhibit electron transfer from iron to the contaminant. Viable cell counts showed that N25 considerably inhibited microbial growth. Furthermore, TSCM demonstrated that S. algae tends to adhere to the surface of N25 suggesting that direct nanoparticle attachment may impact both the microbial population and the electron transfer from the iron to the contaminant. Data suggested that S. algae contributes significantly to N25 anoxic corrosion through microbial iron respiration. Our findings provide better insights about the fate of nZVI in the subsurface and its interactions with surrounding microorganisms.

Low-Frequency Electrochemical Impedance Spectroscopy as a Monitoring Tool for Yeast Growth in Industrial Brewing Processes

Today’s yeast total biomass and viability measurements during the brewing process are dependent on offline methods such as methylene blue or florescence dye-based staining, and/or the usage of flow cytometric measurements. Additionally, microscopic cell counting methods decelerate an easy and quick prediction of yeast viability. These processes are time consuming and result in a time-delayed response signal, which not only reduces the knowledge of the performance of the yeast itself, but also impacts the quality of the final product. Novel approaches in process monitoring during the aerobic and anaerobic fermentation of Saccharomyces cerevisiae are not only limited to classical pH, dO2 and off-gas analysis, but they also use different in situ and online sensors based on different physical principles to determine the biomass, product quality and cell death. Within this contribution, electrochemical impedance spectroscopy (EIS) was used to monitor the biomass produced in aerobic and anaerobic batch cultivation approaches, simulating the propagation and fermentation unit operation of industrial brewing processes. Increases in the double-layer capacitance (CDL), determined at frequencies below 1 kHz, were proportional to the increase of biomass in the batch, which was monitored in the online and inline mode. A good correlation of CDL with the cell density was found. In order to prove the robustness and flexibility of this novel method, different state-of-the-art biomass measurements (dry cell weight—DCW and optical density—OD) were performed for comparison. Because measurements in this frequency range are largely determined by the double-layer region between the electrode and media, rather minor interferences with process parameters (aeration and stirring) were to be expected. It is shown that impedance spectroscopy at low frequencies is not only a powerful tool for the monitoring of viable yeast cell concentrations during operation, but it is also perfectly suited to determining physiological states of the cells, and may facilitate biomass monitoring in the brewing and yeast-propagating industry drastically.

Approaches for Eliminating Bacteria Introduced during &lt;i&gt;In Situ&lt;/i&gt; Bioleaching of Fractured Sulfidic Ores in Deep Subsurface

The major objective of the EU Horizon 2020 project “BioMOre” is the technical realization of indirect in situ leaching of Kupferschiefer sandstone and black shale ore by a ferric iron lixiviant generated by a mixed culture of autotrophic, acidophilic, iron-oxidizing bacteria and archaea in a ferric iron-generating bioreactor (FIGB). These organisms could colonize the deeply buried geological formations even under anaerobic conditions as most are able to grow by coupling the reduction of ferric iron to the oxidation of reduced sulfur compounds in the absence of oxygen. Development of an inhibition protocol to eliminate these allochthonous microbial bioreactor populations subsequent to the completion of in situ bioleaching was therefore investigated. Column bioleaching experiments using a laboratory-scale FIGB confirmed not only that metals were solubilised from both the sandstone and shale ores, but also that significant numbers of bacteria were released from the FIGB. The efficacy of 13 different chemical compounds in inhibiting microbial iron oxidation has been tested at different concentrations in shake flask and FIGB-coupled columns. Iron-oxidation activity, microcalorimetrically-determined activity and ATP measurements, in combination with microscopic cell counts and biomolecular analysis (T-RFLP, qPCR), plate counts and most-probable-number (MPN), were used to monitor the inhibiting effects on the acidophiles. Complete inhibition of metabolic activity of iron-oxidizing acidophiles was achieved in the presence of 0.4 mM formate, 300 mM chloride, 100 mM nitrate, 10 mM of primary C6 to C8 alcohols, 100 mM 1-butanol, 100 mM 1-pentanol, 0.1 mM SDS or 0.35 mM benzoic acid. No inhibition was found for 0.6 mM acetic acid and 200 mM methanol. Based on these results a recipe for the chemical composition of the “decommissioning solution” is proposed.

Effect of X-Ray µCT Scanning on the Growth and Activity of Microorganisms in a Heap Bioleaching System

In heap bioleaching, a process in which microorganisms are required for the regeneration of leach reagents and control of reaction products, inaccessibility of non-surface mineral grains is a key cause of low recovery and long extraction times. High resolution, non-destructive 3D X-ray micro-computed tomography (μCT) is an imaging technique that has been successfully demonstrated for the study of abiotic leaching of non-surface minerals. For this technique to be applied to biotic leaching, it is required that the iron and sulphur oxidizing abilities of the microorganisms are not affected by the irradiation experienced. In the current study, the feasibility of investigating biotic leaching by X-ray μCT is explored by examining the relative energies required to achieve the high image resolution needed for mineral grain mapping while avoiding microbial deactivation. A mixed mesophilic and moderately thermophilic culture in solution was used and exposed to various X-ray energy doses. Direct microscopic cell counting and redox potential were measured to quantify the microbial activity and growth. The results showed that exposure to X-ray does not affect microbial activity at 35-90 kV, 200-280 μA and a distance of 7.2 cm between energy source and sample, however, it has an influence at 120 and 150 kV. This indicates that while X-ray μCT does influence the microbial cultures, it can be used for bioleaching studies at lower energy doses.

Application of a portable microscopic cell counter for the counting of residual leukocytes in leukoreduced apheresis platelet concentrates in a hospital blood bank

While a portable microscopic cell counter has been evaluated to enumerate residual white blood cells (WBCs) in red blood cells and platelet concentrates at blood centers, it has not yet been assessed in a hospital blood bank. We investigated the performance of this device and evaluated its accuracy, along with its benefits in time management.Residual WBCs from each of 100 apheresis platelet specimens were measured manually using a Nageotte chamber, along with flow cytometry methods and an ADAM-rWBC automated instrument (NanoEnTek, Seoul, South Korea). The efficiency was calculated by measuring the time required for the analysis of one specimen ten times consecutively.Flow cytometry and the ADAM-rWBC were able to detect four sporadic cases that had residual WBCs exceeding 1/μL that were not detected by the manual method. Analysis time was the shortest with the ADAM-rWBC, followed by flow cytometry and the manual method.Our data suggest that hospital blood banks require quality control of residual WBCs; among the methods evaluated in this study, the portable microscopic cell counter offers the best time efficiency.

Research on high-accuracy biological microscopic imaging and cell counting system

In this study, an observing method which adopts to tilt observation cells through transparent side-wall of cell factory has been put forward to break through the limitation of monitoring method for cell factory. An photoelectric microscopic imaging system with 85mm working distance, large depth of field and 45 tilt angle has been designed, which can real-time observe each layer cell factory. Meanwhile, according to the characters of the obtained images uneven illumination, sample turbidity, cell overlap and the cell distance, the single scale Retinex (SSR) enhancement algorithm has been adopted to preprocess the image, and then the OTSU is used to segment the image. Finally, a high-accuracy cell counting method based on mathematical statistics and quick connected component labeling is presented. A large number of experimental results show that the resolution of the system can reach 1.1 um, which meets the requirement of resolution and clearance in biological engineered application. Moreover, the cell image processing method has a good visual effect, and the cell counting accuracy is above 93%. In a word, the detection system is suitable for various cells with great significant engineering value.

Transforming growth factor-β (TGF-β) activation in cutaneous wounds after topical application of aloe vera gel.

Aloe vera is a medicinal plant used to treat various skin diseases. The effects of using aloe vera gel on the healing process were investigated by microscopic methods, cell counting, and TGF-β gene expression in the wound bed. Sixty Wistar rats weighing 200-250 g were placed under anesthesia in sterile conditions. A square 1.5 cm × 1.5 cm wound was made on the back of the neck. The rats were divided into control and 2 experimental groups. Additionally, the control and experimental groups were separated into 3 subgroups corresponding to 4, 7, and 14 days of study. In the first experimental group, aloe vera was used twice on the wound. The second experimental group received aloe vera overtreatment once on the wound. The positive control group received daily application of 1% phenytoein cream following surgical wound creation. The control group did not receive any treatment. This tissue was examined using histological staining (H&E) and Masson's Trichrome. Wound surface and wound healing were evaluated separately. TGF-β gene expression was analyzed by RT-PCR. Results showed that fibroblasts in both experimental groups were significantly increased, thereby acceleration wound healing. Application of aloe vera gel will increase TGF-β gene expression, ultimately accelerating the wound healing process.

Elucidation of Taste- and Odor-Producing Bacteria and Toxigenic Cyanobacteria in a Midwestern Drinking Water Supply Reservoir by Shotgun Metagenomic Analysis.

While commonplace in clinical settings, DNA-based assays for identification or enumeration of drinking water pathogens and other biological contaminants remain widely unadopted by the monitoring community. In this study, shotgun metagenomics was used to identify taste-and-odor producers and toxin-producing cyanobacteria over a 2-year period in a drinking water reservoir. The sequencing data implicated several cyanobacteria, including Anabaena spp., Microcystis spp., and an unresolved member of the order Oscillatoriales as the likely principal producers of geosmin, microcystin, and 2-methylisoborneol (MIB), respectively. To further demonstrate this, quantitative PCR (qPCR) assays targeting geosmin-producing Anabaena and microcystin-producing Microcystis were utilized, and these data were fitted using generalized linear models and compared with routine monitoring data, including microscopic cell counts, sonde-based physicochemical analyses, and assays of all inorganic and organic nitrogen and phosphorus forms and fractions. The qPCR assays explained the greatest variation in observed geosmin (adjusted R(2) = 0.71) and microcystin (adjusted R(2) = 0.84) concentrations over the study period, highlighting their potential for routine monitoring applications. The origin of the monoterpene cyclase required for MIB biosynthesis was putatively linked to a periphytic cyanobacterial mat attached to the concrete drinking water inflow structure. We conclude that shotgun metagenomics can be used to identify microbial agents involved in water quality deterioration and to guide PCR assay selection or design for routine monitoring purposes. Finally, we offer estimates of microbial diversity and metagenomic coverage of our data sets for reference to others wishing to apply shotgun metagenomics to other lacustrine systems. Cyanobacterial toxins and microbial taste-and-odor compounds are a growing concern for drinking water utilities reliant upon surface water resources. Specific identification of the microorganism(s) responsible for water quality degradation is often complicated by the presence of co-occurring taxa capable of producing these undesirable metabolites. Here we present a framework for how shotgun metagenomics can be used to definitively identify problematic microorganisms and how these data can guide the development of rapid genetic assays for routine monitoring purposes.

Microscopy cell counting and detection with fully convolutional regression networks

This paper concerns automated cell counting and detection in microscopy images. The approach we take is to use convolutional neural networks (CNNs) to regress a cell spatial density map across the image. This is applicable to situations where traditional single-cell segmentation-based methods do not work well due to cell clumping or overlaps. We make the following contributions: (i) we develop and compare architectures for two fully convolutional regression networks (FCRNs) for this task; (ii) since the networks are fully convolutional, they can predict a density map for an input image of arbitrary size, and we exploit this to improve efficiency by end-to-end training on image patches; (iii) we show that FCRNs trained entirely on synthetic data are able to give excellent predictions on microscopy images from real biological experiments without fine-tuning, and that the performance can be further improved by fine-tuning on these real images. Finally, (iv) by inverting feature representations, we show to what extent the information from an input image has been encoded by feature responses in different layers. We set a new state-of-the-art performance for cell counting on standard synthetic image benchmarks and show that the FCRNs trained entirely with synthetic data can generalise well to real microscopy images both for cell counting and detections for the case of overlapping cells.

Characterization of primary chondrocytes harvested from hips with femoroacetabular impingement

Acetabular chondral lesions are common in patients with femoroacetabular impingement (FAI) syndrome. The aim of this study was (1) to evaluate the proliferation potential of primary human chondrocytes (hC) derived from both acetabular and femoral site and (2) to validate cellular differentiation during three-dimensional (3D) cultivation as a prerequisite for autologous matrix-assisted cartilage regeneration of the hip joint. hC were isolated from cartilage samples obtained from N=6 patients during offset reconstruction. Proteoglycan content was assessed by Safranin-O staining. Proliferation and cell viability were quantified by microscopic cell counting and Trypan Blue exclusion. Messenger ribonucleic acid (mRNA) expression levels of collagen type 1 and 2, aggrecan (ACAN), and interleukin-1β (IL-1β) genes were assessed upon monolayer cultivation, after 48h/4-10°C - transport simulation and after 14 days of 3D hydrogel cultivation. Primary hC from acetabular and femoral damaged sites were viable. No significant intergroup differences were observed concerning cell viability (>95%) after monolayer cultivation and transport simulation. Harvest yields from acetabular and femoral cartilage samples were comparable to that known from knee joints (mean±standard deviation (SD), 13.4×10(6)±5×10(6) cells per culture vs 20×10(6) cells). Redifferentiation was induced during 3D hydrogel cultivation as observed by increased levels of collagen II (1000-fold) and ACAN (10-fold) gene vs monolayer cultivation (P<0.001). hC derived from damaged acetabular and femoral site are qualified for autologous matrix-assisted cartilage transplantation paving the way for cell-based cartilage regeneration in FAI patients.

Demonstration of a multi-technique approach to assess glacial microbial populations in the field

ABSTRACTThe ability to perform microbial detection and characterization in-field at extreme environments, rather than on returned samples, has the potential to improve the efficiency, relevance and quantity of data from field campaigns. To date, few examples of this approach have been reported. Therefore, we demonstrate that the approach is feasible in subglacial environments by deploying four techniques for microbial detection: real-time polymerase chain reaction; microscopic fluorescence cell counts, adenosine triphosphate bioluminescence assay and recombinant Factor C assay (to detect lipopolysaccharide). Each technique was applied to 12 subglacial ice samples, 12 meltwater samples and two snow samples from Engabreen, Northern Norway. Using this multi-technique approach, the detected biomarker levels were as expected, being highest in debris-rich subglacial ice, moderate in glacial meltwater and low in clean ice (debris-poor) and snow. Principal component analysis was applied to the resulting dataset and could be performed in-field to rapidly aid the allocation of resources for further sample analysis. We anticipate that in-field data collection will allow for multiple rounds of sampling, analysis, interpretation and refinement within a single field campaign, resulting in the collection of larger and more appropriate datasets, ultimately with more efficient science return.

Comparison of four measuring techniques to assess growth inhibition in standardized tests with seven freshwater algae and cyanobacteria

ABSTRACTAs representatives of primary producers, algae and cyanobacteria play an important role in assessing the risk of chemicals. However, the list of standard algal species commonly used for toxicity testing comprises very few species so that there is an urgent demand to identify further non-standard species which can be used for higher-tier risk assessment in general and the analysis of species sensitivity distributions in particular. In this study, four measuring techniques to assess growth inhibition were compared using five non-standard and two standard algal species and 3,5-dichlorophenol as reference substance. Regarding sensitivity and suitability, the measuring techniques were ranked in the following descending order: delayed fluorescence, prompt fluorescence, photometric absorbance, and microscopic cell count. All five non-standard species fulfilled the validity criteria if tested with delayed fluorescence.