Microscopic Enumeration Research Articles

Effect of water temperature on the signal of a phycocyanin probe and correction factor applied to three morphotypes to improve field monitoring of cyanobacteria

In a context of global warming and heavy anthropization of freshwater systems, cyanobacteria monitoring is becoming an essential priority. In recent years, submersible fluorescence sensors capable of measuring pigments and estimating cyanobacterial biomass in near-real time have been deployed on a large-scale. However, these tools have several limitations, such as the detection limit of pigments like phycocyanin or the impact of temperature on the measurements. In an attempt to overcome these shortcomings, we compared the effectiveness of a phycocyanin probe at different temperatures, first on three cyanobacteria cultures (Microcystis, Dolichospermum and Synechocystis) and then on a small eutrophic lake in continuous recording for three years.In vitro results showed a strong correlation between cyanobacterial biomasses and phycocyanin probe measurements, with R2 higher than 0.96, even for biovolumes lower than hundreds of mm3.L−1. A saturation effect was demonstrated between 350 and 550 mm3.L−1, depending on the strain. In addition, laboratory tests on temperature highlighted the sensor's limitations, with a strong over- and underestimation at low (< 15 °C) and high (> 25 °C) temperatures, respectively. Given this discrepancy, a water temperature correction factor was determined for each strain and applied according to the predominant cyanobacteria group identified in the field during the three years monitoring. Signal quality improved considerably, and the stronger relationship found between the corrected signal and the biovolume of cyanobacteria obtained by microscopic enumeration enabled us to validate this temperature correction factor, offering the possibility of using it to better monitor cyanobacterial dynamics.

Effect of micro-plastic particles on coral reef foraminifera

Foraminifera are single-celled protists which are important mediators of the marine carbon cycle. In our study, we explored the potential impact of polystyrene (PS) microplastic particles on two symbiont-bearing large benthic foraminifera species—Heterostegina depressa and Amphistegina lobifera—over a period of three weeks, employing three different approaches: investigating (1) stable isotope (SI) incorporation—via 13C- and 15N-labelled substrates—of the foraminifera to assess their metabolic activity, (2) photosynthetic efficiency of the symbiotic diatoms using imaging PAM fluorometry, and (3) microscopic enumeration of accumulation of PS microplastic particles inside the foraminiferal test. The active feeder A. lobifera incorporated significantly more PS particles inside the cytoplasm than the non-feeding H. depressa, the latter accumulating the beads on the test surface. Photosynthetic area of the symbionts tended to decrease in the presence of microplastic particles in both species, suggesting that the foraminiferal host cells started to digest their diatom symbionts. Compared to the control, the presence of microplastic particles lead to reduced SI uptake in A. lobifera, which indicates inhibition of inorganic carbon and nitrogen assimilation. Competition for particulate food uptake was demonstrated between algae and microplastic particles of similar size. Based on our results, both species seem to be sensitive to microplastic pollution, with non-feeding H. depressa being more strongly affected.

Comparison Evaluation of Automated Nucleated Red Blood Cell Enumeration by Sysmex XN 1000 in Comparison With Microscopic Reference in Children Under 1 Year.

In newborns, elevated nucleated red blood cell (NRBC) levels can be associated with enhanced erythropoietic stress and might be predictive for adverse outcome. Also, the presence of NRBC in peripheral blood might lead to erroneous enumeration results of white blood cells in automated hematology analyzers. We aimed to assess the comparability of the Sysmex XN 1000 to manual slide reviews and correlation of NRBC with inflammation markers. Specimens of 3397 children under 1 year were compared by automated and microscopic NRBC enumeration. Additionally, potential correlations between NRBC and age and inflammation markers were examined. Overall, there was good correlation (r = 0.97) between automated (range: 0%-3883%) and microscopic enumeration (range: 0%-3694%) of NRBC with high comparability up to a NRBC value of 200% and an increase in the variation between the two methods with increasing NRBC numbers. When 94 samples with ≤ 200% NRBC and ≥ 30% divergence between methods were separately reanalyzed with respect to overlapping cell populations in their scattergrams, Sysmex would have generated unrecognized incorrect automated results in 47 samples, corresponding to 1.4% of total study samples. NRBC counts were negatively correlated to age, but not to inflammation markers. Sysmex XN 1000 is highly precise in the enumeration of NRBC in children under 1 year up to counts of 200% and might replace time-intense manual counting in routine diagnostics. In the setting of neonatal and intensive care diagnostics, microscopic control and supervision of scattergrams are highly recommended for any automated NRBC enumeration processes.

Morphokinetic changes in vitrified and non-vitrified in vitro-derived ovine embryos

The present experiment employed time-lapse (TL) imaging to assess the effects of vitrification on the development of ovine blastocysts and to see if the timing of blastocyst formation and expansion was correlated with the numbers of embryo- and trophoblasts determined through differential staining of the expanded blastocysts. Ovaries were obtained after slaughter from cycling (October–March) Polish Longwool ewes aged 1–3 years and cumulus-oocyte complexes were collected by ovarian scarification. In vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10% of FBS, and 5 μg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were incubated with thawed ram semen (IVF) for 19 h and all presumptive zygotes were transferred to a 16-well dish containing Cult medium for monitoring with the Primo Vision TL system. A portion of ovine embryos were vitrified (Cryotop system) at the early cavitation stage and TL observations of warmed (n = 30) and non-vitrified (n = 32) blastocysts continued until the attainment of the expanded blastocyst stage, at which point they were differentially stained with bisbenzimide and propidium iodide for microscopic enumeration of embryoblasts (inner cell mass blastomeres) and trophoblast-cells. There were no statistically significant differences in the timing of blastulation (tB) and formation of expanded blastocysts (tEB) between vitrified and non-vitrified ovine embryos, but non-vitrified blastocysts exceeded (P < 0.05) their vitrified and warmed counterparts in the mean number of embryo- and trophoblasts. In addition, the number of trophoblasts was negatively and moderately correlated with tB and tEB, for both vitrified and non-vitrified embryos. It can be concluded that even though vitrification of ovine embryos is associated with a significant reduction in the number of blastomeres, the rate of blastocyst development remains closely linked to the numbers of trophoblastic cells.

Chlorophyll and phycocyanin in-situ fluorescence in mixed cyanobacterial species assemblages: Effects of morphology, cell size and growth phase

Cyanobacteria harmful blooms can represent a major risk for public health due to potential release of toxins and other noxious compounds in the water. A continuous and high-resolution monitoring of the cyanobacteria population is required due to their rapid dynamics, which has been increasingly done using in-situ fluorescence of phycocyanin (f-PC) and chlorophyll a (f-Chl a). Appropriate in-situ fluorometers calibration is essential because f-PC and f-Chl a are affected by biotic and abiotic factors, including species composition. Measurement of f-PC and f-Chl a in mixed species assemblages during different growth phases - representative of most field conditions - has received little attention. We hypothesized that f-PC and f-Chl a of mixed assemblages of cyanobacteria may be accurately estimated if taxa composition and fluorescence characteristics are known. We also hypothesized that species with different morphologies would have different fluorescence per unit cell and biomass. We tested these hypotheses in a controlled culture experiment in which photosynthetic pigment fluorescence, chemical pigment extraction, optical density and microscopic enumeration of four common cyanobacteria species (Aphanocapsa sp, Microcystis aeruginosa, Dolichospermum circinale and Raphidiopsis raciborskii) were quantified. Both monocultures and mixed cultures were monitored from exponential to late stationary growth phases. The sum of fluorescence of individual species calculated for mixed samples was not significantly different than measured fluorescence of mixed cultures. Estimated and measured f-PC and f-Chl a of mixed cultures had higher correlations and smaller absolute median errors when estimations were based on fluorescence per biomass instead of fluorescence per cell. Largest errors were overestimations of measured fluorescence for species with different morphologies. Fluorescence per cell was significantly different among most species, while fluorescence per unit biomass was not, indicating that conversion of fluorescence to biomass reduces species-specific bias. This study presents new information on the effect of species composition on cyanobacteria fluorescence. Best practices of deployment and operation of fluorometers, and data-driven models supporting in-situ fluorometers calibration are discussed as suitable solutions to minimize taxa-specific bias in fluorescence estimates.

A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection oflive Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify liveL. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire's disease.

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR.

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

Cytology reveals diverse cell morphotypes and cellin-cell interactions in normal collector sea urchins Tripneustes gratilla.

Echinoderms such as sea urchins are important in marine ecosystems, particularly as grazers, and unhealthy sea urchins can have important ecological implications. For instance, unexplained mortalities of Diadema antillarum in the Caribbean were followed by algal overgrowth and subsequent collapse of coral reef ecosystems. Unfortunately, few tools exist to evaluate echinoderm health, making management of mortalities or other health issues problematic. Hematology is often used to assess health in many animal groups, including invertebrates, but is seldom applied to echinoderms. We used a standard gravitometric technique to concentrate fixed coelomocytes from the collector sea urchin Tripneustes gratilla onto microscope slides, permitting staining and enumeration. Using Romanowsky stain and electron microscopy to visualize cell details, we found that urchin cells could be partitioned into different morphotypes. Specifically, we enumerated phagocytes, phagocytes with perinuclear cytoplasmic dots, vibratile cells, colorless spherule cells, red spherule cells, and red spherule cells with pink granules. We also saw cell-in-cell interactions characterized by phagocytes apparently phagocytizing mainly the motile cells including red spherule cells, colorless spherule cells, and vibratile cells disproportionate to underlying populations of circulating cells. Cell-in-cell interactions were seen in 71% of sea urchins, but comprised <1% of circulating cells. Finally, about 40% of sea urchins had circulating phagocytes that were apparently phagocytizing spicules. The coelomic fluid collection and slide preparation methods described here are simple, field portable, and might be a useful complementary tool for assessing health of other marine invertebrates, revealing heretofore unknown physiological phenomena in this animal group.

Microbial Assessment of Grey Water Samples Treated with Activated Carbon Forms of Selected Agro-wastes

This study evaluated the efficacy of activated carbon from rice husk, corn cob and coconut husk wastes in the reduction of microbial properties of grey water samples harvested from students’ hostels. Microscopic characterization, enumerations and identification of microbial isolates were carried out to determine the microbial community before and after the treatment with activated carbon. Staphylococcus sp, Micrococcus sp, Bacillus sp, Salmonella sp, Saccharomyces sp and Penicillium sp were observed to be present in the grey water. Before treatment, Total Heterotrophic Count (THC) was 1.2 x 1011cfu/ml, Total Coliform Count (TCC), 6.4 x 106cfu/ml and Total Fungi Count (TFC) 2.2 x 1010cfu/ml. THC after the treatment ranged from 1.69 x 109 - 7.6 x 1010cfu/ml; TCC, 2.2 x 105 - 7.3 x 108cfu/ml and TFC 1.0 x 108 - 1.2 x 109cfu/ml. Reduction in the microbial load after treatment revealed that activated carbons from rice husk, corn cob and coconut husk can be used singly or in combined states for the treatment of wastewater.

Spatial distribution of microplastics around an inhabited coral island in the Maldives, Indian Ocean

Small plastic particles are considered environmental pollutants and are highly concentrated in marine sediments. However, knowledge about plastic abundance within coral reef habitat and beach sediments surrounding remote inhabited coral islands is scarce. In this study, microplastic accumulation was investigated on a small inhabited coral island located in the Maldives. Sediments from 22 sampling sites across fore reef, reef flat, and beach environments were analysed for plastic particles <5 mm. Density separation and microscope enumeration revealed a total of 1244 individual microplastic pieces, in filamentous (49%) and fragmented (51%) forms, found across all sampling sites. High concentrations were recorded at all sites, however, there was no significant relationship between microplastic concentration or size across regions (inner atoll and outer atoll) or environments (fore reef, reef flat, and beach). Furthermore, concentrations of microplastic fragment and filament forms, total concentration, and the microplastic community, showed little correlation with sediment particle size. Our findings show microplastics are ubiquitous in marine sediments around a remote coral island, at sizes ingestible by marine organisms, raising concerns about potential effects of microplastic ingestion by coral reef species.

Rapid shifts in methanotrophic bacterial communities mitigate methane emissions from a tropical hydropower reservoir and its downstream river

Methane-oxidizing bacteria (MOB) present in the water column mitigate methane (CH4) emissions from hydropower complexes to the atmosphere. By creating a discontinuity in rivers, dams cause large environmental variations, including in CH4 and oxygen concentrations, between upstream, reservoir, and downstream segments. Although highest freshwater methanotrophic activity is often detected at low oxygen concentrations, CH4 oxidation in well-oxygenated downstream rivers below dams has also been reported. Here we combined DNA and RNA high-throughput sequencing with microscopic enumeration (by CARD-FISH) and biogeochemical data to investigate the abundance, composition, and potential activity of MOB taxa from upstream to downstream waters in the tropical hydropower complex Batang Ai (Malaysia). High relative abundance of MOB (up to 61% in 16S rRNA sequences and 19% in cell counts) and enrichment of stable isotopic signatures of CH4 (up to 0‰) were detected in the hypoxic hypolimnion of the reservoir and in the outflowing downstream river. MOB community shifts along the river-reservoir system reflected environmental sorting of taxa and an interrupted hydrologic connectivity in which downstream MOB communities resembled reservoir's hypolimnetic communities but differed from upstream and surface reservoir communities. In downstream waters, CH4 oxidation was accompanied by fast cell growth of particular MOB taxa. Our results suggest that rapid shifts in active MOB communities allow the mitigation of CH4 emissions from different zones of hydropower complexes, including in quickly re-oxygenated rivers downstream of dams.

Analytical validation of iSort digital cytometry for leukocyte enumeration in clinical tumor specimens.

e15243 Background: Tumor infiltrating leukocytes (TILs) are major determinants of anti-tumor immune responses and mediate effector functions for diverse immunotherapies. Techniques relying on intact cells such as flow cytometry (FACS), immunohistochemistry (IHC), and scRNA-Seq can be powerful for measuring TIL heterogeneity. However, such methods are often impractical on large patient cohorts, can be poorly quantitative, and some cannot be applied to formalin-fixed paraffin embedded (FFPE) specimens. We developed iSort, a transcriptome deconvolution method based on CIBERSORTx for TIL enumeration. We evaluated iSort by comparing its performance in FFPE and fresh frozen (FF) human tumor tissues to established methods. Methods: We profiled human tumors for diverse TIL subsets, as quantified by iSort, FACS, and IHC. iSort was performed on RNA-Seq libraries derived from bulk human tumors, including FF tumor tissue samples, tumor cell suspensions obtained after dissociation, and FFPE specimens. We assessed the influence of tissue fixation on common TIL subsets enumerated by iSort, by comparing matched pairs of FF and FFPE lung tumor specimens (NSCLC). Finally, we compared iSort with automated microscopic enumeration of CD8 TILs by IHC on primary human lung tumors (FFPE/NSCLC). Results: iSort TIL enumeration was highly correlated with FACS across primary human tumors (CRC = 3, HNSCC = 1) for common TIL subsets (B cells, CD8 T, CD4 T, monocytes/macrophages, dendritic cells, PMNs/MDSCs) and total immune content (median r = 0.91 [0.71 - 0.99]). Tissue dissociation caused significant depletion of monocytes/macrophages (p &lt; 0.01), when comparing bulk tumor specimens with cell suspensions after sample digestion. iSort showed high correlation across 12 TIL subsets when comparing 9 pairs of FF and FFPE specimens (r = 0.90). Finally, we observed high linearity between iSort and microscopic enumeration of CD8 TILs from IHC (r = 0.87, n = 5). Conclusions: iSort digital cytometry achieves accurate and robust TIL enumeration as compared with FACS and IHC, showing comparable performance in fixed and frozen primary human tumors. Importantly, iSort is directly applicable to bulk tumors and is unaffected by tissue dissociation artifacts, unlike methods requiring cell suspensions for TIL enumeration (FACS, scRNA-seq). Therefore, iSort is a promising approach for development and measurement of immunotherapy biomarkers.

A fully integrated bacterial pathogen detection system based on count-on-a-cartridge platform for rapid, ultrasensitive, highly accurate and culture-free assay

Rapid, sensitive and accurate point-of-care-testing (POCT) of bacterial load from a variety of samples can help prevent human infections caused by pathogenic bacteria and mitigate their spreading. However, there is an unmet demand for a POCT device that can detect extremely low concentrations of bacteria in raw samples. Herein, we introduce the ‘count-on-a-cartridge’ (COC) platform for quantitation of the food-borne pathogenic bacteria Staphylococcus aureus. The system comprised of magnetic concentrator, sensing cartridge and fluorescent image reader with a built-in counting algorithm facilitated fluorescent microscopic bacterial enumeration in user-convenient manner with high sensitivity and accuracy within a couple of hours. The analytical performance of this assay is comparable to that of a standard plate count. The COC assay shows a sensitivity of 92.9% and specificity of 100% performed according to global microbiological criteria for S. aureus which is acceptable below 100 CFU/g in the food matrix. This culture-independent, rapid, ultrasensitive and highly accurate COC assay has great potential for places where prompt bacteria surveillance is in high demand.

Albumin and Globulin Fractions from Black Bean Seeds (Phaseolus vulgaris L.) used in the Management of Sickle Cell Disease (SCD) in the West Region of Cameroon have Antisickling and Antioxidant Properties

Background: Peptides have shown anti-sickling effect and are able to reduce oxidative stress, thus contributing to the management of sickle cell disease. Objectives: The present study was undertaken to characterize and to assess the antisickling and antioxidant properties of albumins and globulins fractions from black bean seed (Phaseolus vulgaris L.) used to manage sickle cell disease in West Region of Cameroon. Methods: NaCl 5% (1:10 w/v) and dialysis were used for extracting albumins and globulins respectively. The concentration of proteins was determined by the Bradford method. Molecular size was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In vitro antisickling properties, investigate was carried up using microscopic enumeration. Membrane stability effect and antioxidant potentials were determined using colorimetric method. Results: In non-reducing buffer showed both albumin and globulin in native form, had the main proteins with dense band of about 130 and 37 kDa. Antisickling tests revealed the best inhibition (79.15 ± 3%) and reversibility (73.49 ± 6%) rates with globulin fraction at 3.82 mg/ml. Hemolysis decreased significantly with albumin and globulin fractions at different concentrations showing the stability effect on the membranes of erythrocytes. However the concentration of globulin at 3.82 mg/ml has shown the best activity (from 100 to 4.62%). The FRAP test has shown that the proteins extracted from black bean seeds (Phaseolus vulgaris L.) have a global antioxidant power of 48.38 ± 5 mg FeII/100g (albumin fraction) and 7.26 ± 4 mg Fell/100g (globulin fraction). The value obtained from the albumin fraction was significantly lower (p<0.05) than that obtained with gallic acid used as standard. However, the albumin fraction showed better anti-free radical activity on •DPPH (IC50 =7.37 ± 0.2 mg/ml) and the hydroxyl radical (IC50 = 5.96 ± 0.1 mg/ml) than the globulin fraction (IC50 =13.56 ± 0.8 mg/ml for the •DPPH radical and 13.41 ± 2 mg/ml for the •OH radical. Conclusion: Albumins and globulins from black bean seeds used to manage sickle cell disease in the west region of Cameroon have antisickling, anti-hemolytic and antioxidant properties. These results may justify the use of black bean seeds by sickle cell patients.

Algicidal bacterium CZBC1 inhibits the growth of Oscillatoria chlorina, Oscillatoria tenuis, and Oscillatoria planctonica

Frequent harmful cyanobacteria blooms limit the sustainable development of aquaculture. Algicidal bacteria can efficiently control harmful algae without secondary pollution. The algicidal bacteria CZBC1 can lyse Oscillatoria spp. and other harmful cyanobacteria, but its effector mechanism and algicidal threshold are unknown. In this study, we examined the algicidal effect of CZBC1 on O. chlorina, O. tenuis, and O. planctonica by microscopic enumeration and scanning electron microscopy observation. Then, we examined the alginolytic effects of CZBC1 (concentrations 103–106 colony forming units (cfu)/mL) on these three species (concentrations 103–106 cells/mL) to determine the effective concentrations of CZBC1 for Oscillatoria spp. alginolysis. Results showed that CZBC1 can directly lyse O. chlorina and O. tenuis but indirectly lyse O. planctonica. When the initial concentration of CZBC1 was 106 cfu/mL, alginolytic effects were high for all three species at all concentrations, and the alginolytic rate could reach 100% in 3–9 days. When the initial concentration of CZBC1 was lower (103 cfu/mL), its inhibitory effects were delayed by 2–5 days, but the cell counts were significantly decreased compared with the control, evidencing significant alginolysis. In addition, the higher the concentration of the algicidal bacteria suspension, the more significant the alginolytic effects. Our results indicate that CZBC1 has different alginolytic mechanisms for O. chlorina, O. tenuis, and O. planctonica, and that different initial concentrations of CZBC1 have different alginolytic effects on these algal species.

Quantification of cell concentration in biofuel-important microalgae using hyperspectral reflectance and hyperspectral extinction coefficient

ABSTRACT Monitoring of microalgae cell concentration during their growing phase is imperative to ensure efficiency in biomass production and to study the cell division kinetics since it impacts the penetration depth of the active light radiation and thus determines the thickness of the active growing layer. Conventionally, the cell concentration (number of cells per unit volume) of microalgal solutions is estimated by microscopic enumeration method that is laborious and time consuming and can be performed only in the laboratory. In this study, we developed algorithms that relate cell concentration to hyperspectral reflectance and extinction coefficient (EC) for quick estimates of cell concentration. A multi-layer radiative transfer model was developed to correct the effect of the bottom of the microalgal solution container and table surface to obtain the hyperspectral reflectance and EC of only microalgal solution. Regression results show that the reflectance-based Band Ratio (BR) algorithm and the EC-based Spectral Shape (SS) index in the near-infrared (NIR) band gave the best results for all three microalgae species with the coefficient of determination , mean relative errors MRE < 5% and root mean squared error RMSE < 5%, and especially for A. cylindrica and N. gaditana, with , MRE < 2% and RMSE < 1%, respectively. These relationships can be used to quickly estimate microalgal cell concentration from hyperspectral measurements that can be carried out quickly and easily either in laboratory or in field.

An aptasensor based on the microscopic enumeration of encoding gold nanoparticles for the detection of C-reactive protein

C-reactive protein (CRP) is a crucial clinical biomarker for inflammatory and cardiovascular diseases. Therefore, the sensitive, selective and convenient detection of CRP is of great significance. Using gold nanoparticles (AuNPs) and combining the specific interaction between an aptamer and CRP, we developed a simple and convenient assay for CRP detection. The aptamer-based probe was fabricated through the hybridization of CRP-aptamer immobilized on magnetic beads (MBs) to a short complementary DNA (cDNA) chain attached to AuNPs to form a MB–Aptamer–AuNP sandwich structure. Upon the addition of CRP, aptamer–cDNA dehybridization occurred due to the strong interaction between CRP and the aptamer, resulting in the release of AuNPs, which were subjected to DFM imaging and subsequently counted using the MATLAB program. The number of AuNPs was therefore positively correlated to the concentration of CRP and a detection limit as low as 2.71 nM was achieved. The current approach could also exclude the disturbance of other proteins, including thrombin, IgG, Lys and BSA. In addition, the concentration of CRP detected was in good agreement with the amount cast in bovine and mouse serum, indicating that the proposed probe is robust and accurate, and it is very promising for practical applications where CRP detection is necessary. The current strategy is also promising for the detection of other proteins where a suitable aptamer is selected.

Cross-over data supporting long-term antibiotic treatment in patients with painful lower urinary tract symptoms, pyuria and negative urinalysis

PurposeTo measure the effects of an unplanned, sudden cessation of treatment in an unselected group of patients with chronic painful LUTS managed with protracted antimicrobial treatment and to report these observational data collected from a cross-over process.Materials and methodsThe imposition of a guideline resulted in the immediate cessation of antibiotic treatment in a cohort of patients with chronic painful LUTS and microscopic pyuria. Patients were assessed before treatment withdrawal, whilst off treatment, and following reinstatement. Outcome measures included a validated symptom score, microscopic enumeration of urinary white cells and uroepithelial cells, and routine urine culture.ResultsThese patients had reported treatment-resistant, painful LUTS for a mean of 6.5 years before treatment at this centre. Treatment was stopped in 221 patients (female = 210; male = 11; mean age = 56 years; SD = 17.81). Sixty-six per cent of women were post-menopausal. After unplanned treatment cessation, 199 patients (90%; female = 188; male = 9) reported deterioration. Eleven patients required hospital care in association with disease recurrence, including acute urinary tract infection (UTI) and urosepsis. Symptom scores increased after cessation and recovered on reinitiating treatment (F = 33; df = 2; p < 0.001). Urinary leucocyte (F = 3.7; df = 2; p = 0.026) and urothelial cells counts mirrored symptomatic changes (F = 6.0; df = 2; p = 0.003). Routine urine culture results did not reflect changes in disease status.ConclusionThese data support the hypothesis that treating painful LUTS associated with pyuria with long-term antimicrobial courses, despite negative urine culture, is effective. The microscopy of fresh unspun, unstained urine to count white cells and epithelial cells offers a valid method of monitoring disease. An unplanned cessation of antibiotic therapy produced a resurgence of symptoms and lower urinary tract inflammation in patients with chronic LUTS, supporting an infective aetiology below the level of routine detection.

Cyanotoxins and Cyanobacteria Cell Accumulations in Drinking Water Treatment Plants with a Low Risk of Bloom Formation at the Source.

Toxic cyanobacteria have been shown to accumulate in drinking water treatment plants that are susceptible to algal blooms. However, the risk for plants that do not experience algal blooms, but that receive a low influx of cells, is not well known. This study determined the extent of cell accumulation and presence of cyanotoxins across the treatment trains of four plants in the Great Lakes region. Samples were collected for microscopic enumeration and enzyme-linked immunosorbent assay (ELISA) measurements for microcystins, anatoxin-a, saxitoxin, cylindrospermopsin, and β-methylamino-L-alanine (BMAA). Low cell influxes (under 1000 cells/mL) resulted in significant cell accumulations (over 1 × 105 cells/mL) in clarifier sludge and filter backwash samples. Microcystins peaked at 7.2 µg/L in one clarifier sludge sample, exceeding the raw water concentration by a factor of 12. Anatoxin-a was detected in the finished drinking water of one plant at 0.6 µg/L. BMAA may have been detected in three finished water samples, though inconsistencies among the BMAA ELISAs call these results into question. In summary, the results show that plants receiving a low influx of cells can be at risk of toxic cyanobacterial accumulation, and therefore, the absence of a bloom at the source does not indicate the absence of risk.

Co-culture models illustrate the digestion of Gemmata spp. by phagocytes

Gemmata spp. bacteria thrive in the same aquatic environments as free-living amoebae. DNA-based detection of Gemmata spp. sequences in the microbiota of the human digestive tract and blood further questioned the susceptibility of Gemmata spp. to phagocytes. Here, Gemmata obscuriglobus and Gemmata massiliana were co-cultured with the amoebae Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba griffini and THP-1 macrophage-like phagocytes. All experiments were performed in five independant replicates. The ratio amoeba/bacteria was 1:20 and the ratio THP-1/bacteria was 1:10. After a 2-hour co-culture, extracellular bacteria were killed by kanamycin or amikacin and eliminated. The intracellular location of Gemmata bacteria was specified by confocal microscopy. Microscopic enumerations and culture-based enumerations of colony-forming units were performed at T = 0, 1, 2, 3, 4, 8, 16, 24, 48 and 72 hours post-infection. Then, Gemmata bacteria were engulfed into the phagocytes’ cytoplasmic vacuoles, more than (98 ± 2)% of Gemmata bacteria, compared to controls, were destroyed by phagocytic cells after a 48-h co-culture according to microscopy and culture results, and no positive culture was observed at T = 72-hours. Under our co-culture conditions, Gemmata bacteria were therefore susceptible to the environmental and host phagocytes here investigated. These data suggest that these Acanthamoeba species and THP-1 cells cannot be used to isolate G. massiliana and G. obscuriglobus under the co-culture conditions applied in this study. Although the THP-1 response can point towards potential responses that might occur in vivo, these responses should first bevalidated by in vivo studies to draw definite conclusions.