Microscopic Counts Research Articles

Quantification of Moss-Associated Cyanobacteria Using Phycocyanin Pigment Extraction.

In the boreal forest, cyanobacteria can establish associations with feather moss and realize the biological nitrogen fixation (BNF) reaction, consisting in the reduction of atmospheric dinitrogen into bioavailable ammonium. In this ecosystem, moss-associated cyanobacteria are the main contributors to BNF by contributing up to 50% of new N input. Current environmental changes driven by anthropogenic activities will likely affect cyanobacteria activity (i.e., BNF) and populations inhabiting mosses, leading to potential important consequences for the boreal forest. Several methods are available to efficiently measure BNF activity, but quantifying cyanobacteria biomass associated with moss is challenging because of the difficulty to separate bacteria colonies from the host plant. Attempts to separate cyanobacteria by shaking or sonicating in water were shown to be poorly efficient and repeatable. The techniques commonly used, microscopic counting and quantitative PCR (qPCR) are laborious and time-consuming. In aquatic and marine ecosystems, phycocyanin (PC), a photosynthesis pigment produced by cyanobacteria, is commonly used to monitor cyanobacteria biomass. In this study, we tested if PC extraction and quantification can be used to estimate cyanobacteria quantity inhabiting moss. We report that phycocyanin can be easily extracted from moss by freeze/thaw disturbance of cyanobacteria cells and can be quickly and efficiently measured by spectrofluorometry. We also report that phycocyanin extraction is efficient (high recovery), repeatable (relative SD < 13%) and that no significant matrix effects were observed. As for aquatic systems, the main limitation of cyanobacteria quantification using phycocyanin is the difference of cellular phycocyanin content between cyanobacteria strains, suggesting that quantification can be impacted by cyanobacteria community composition. Nonetheless, we conclude that phycocyanin extraction and quantification is an easy, rapid, and efficient tool to estimate moss-associated cyanobacteria number.

The Mechanism of the Effect of Exercise Training on the Morphology of Neurons in the Caudate Putamen of the Mice

The purpose of this article is to study the effect of exercise training on neuronal morphology of somatosensory regions and caudate putamen in the cerebral cortex of mice. In this paper, 20 female mice of Kunming species and 20 mice were paired and randomly assigned to the exercise group and the control group. The exercise group mice performed physical activities. After 8 weeks of exercise training on the exercise treadmill, in a quiet state, tissue sections of the somatosensory area of the cerebral cortex were prepared and HE stained. The changes in tissue morphology were observed under an optical microscope and cell counts were performed. The mice were sacrificed during sexual maturity and senescence, and seven morphological parameters in the frontal cortex neurons of two types of mice were measured by cytomorphometry. The test was used to compare the differences between the parameters of different groups. To a certain extent, it can promote the increase of 12% in the nucleolus of canine pyramidal cells in the sensory area of the cerebral cortex of the white mice, and the increase in the density of the spinal cyst spines in the middle pyramidal cells of the sixth layer and the middle astrocytes of the caudal shell. The conclusion shows that proper exercise can increase the activity of neurons in the caudate putamen of the rat brain, which is beneficial to the transmission of neurons and the release of transmitters, and promotes the function of the brain.

Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplexed droplet loop-mediated isothermal amplification (multiplexed dLAMP) with scorpion-shaped probes (SPs) and fluorescence microscopic counting for simultaneous quantification of multiple targets. A set of target-specific fluorescence-activable SPs are designed, which allows establishment of a novel multiplexed LAMP strategy for simultaneous detection of multiple cDNA targets. The digital multiplexed LAMP assay is thus developed by implementing the LAMP reaction using a droplet microfluidic chip coupled to a droplet counting microwell chip. The droplet counting system allows rapid and accurate counting of the numbers of total droplets and the positive droplets by collecting multi-color fluorescence images of the droplets in a microwell. The multiplexed dLAMP assay was successfully demonstrated for the quantification of HCV and HIV cDNA with high precision and detection limits as low as 4 copies per reaction. We also verified its potential for simultaneous digital assay of HCV and HIV RNA in clinical plasma samples. This multiplexed dLAMP technique can afford a useful platform for highly sensitive and specific detection of nucleic acids of viruses and other pathogens, enabling rapid diagnosis and prevention of infectious diseases.

An innovative evaluation method based on polymer mass detection to evaluate the contribution of microfibers from laundry process to municipal wastewater

Clothes washing releases numerous microfibers, including microplastic fibers (MPFs). Although MPFs in laundry wastewater are an important source of microplastics (MPs) in wastewater treatment plants (WWTPs), credible quantitative assessments of their contributions remain limited. Polyester fiber is the most important textile fiber. Its component, polyethylene terephthalate (PET) polymer, can be quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The release of MPFs from polyester clothes through washing was quantified via simulation experiments, and the MPFs in two WWTPs were measured by microscopic counting and LC-MS/MS. Direct comparison of the abundances of PET MPFs in laundry wastewater and WWTP influents led to an undervalued contribution rate of 9%−11% of the PET MPFs in laundry wastewater to those in WWTP influents. However, comparison of the mass of PET MPFs in laundry wastewater and WWTPs influents revealed that the PET MPFs from laundry contributed approximately 50% of those in the WWTPs. The latter was confirmed by comparing the number of polyester fibers released during clothes washing to the calculated number of “model MPFs” in WWTPs according to the PET mass concentration. Based on the PET concentration, the annual discharge of PET MPs from WWTPs to the water environment could also be estimated.

New insights into lake responses to rapid climate change: the Younger Dryas in Lake Gościąż, central Poland

The sediment profile from Lake Gościąż in central Poland comprises a continuous, seasonally resolved and exceptionally well‐preserved archive of the Younger Dryas (YD) climate variation. This provides a unique opportunity for detailed investigation of lake system responses during periods of rapid climate cooling (YD onset) and warming (YD termination). The new varve record of Lake Gościąż presented here spans 1662 years from the late Allerød (AL) to the early Preboreal (PB). Microscopic varve counting provides an independent chronology with a YD duration of 1149+14/–22 years, which confirms previous results of 1140±40 years. We link stable oxygen isotopes and chironomid‐based air temperature reconstructions with the response of various geochemical and varve microfacies proxies especially focusing on the onset and termination of the YD. Cooling at the YD onset lasted ~180 years, which is about a century longer than the terminal warming that was completed in ~70 years. During the AL/YD transition, environmental proxy data lagged the onset of cooling by ~90 years and revealed an increase of lake productivity and internal lake re‐suspension as well as slightly higher detrital sediment input. In contrast, rapid warming and environmental changes during the YD/PB transition occurred simultaneously. However, initial changes such as declining diatom deposition and detrital input occurred already a few centuries before the rapid warming at the YD/PB transition. These environmental changes likely reflect a gradual increase in summer air temperatures already during the YD. Our data indicate complex and differing environmental responses to the major climate changes related to the YD, which involve different proxy sensitivities and threshold processes.

Triggering cultured human osteoblast-like cells’ maturation by an extremely low magnitude alternating electromagnetic field

Introduction: Alternating and pulsed electromagnetic magnetic fields (AEMF and PEMF) of different amplitudes and frequencies can induce metabolic and proliferative effects in osteoblasts, but there is no clearly directed tendency of these effects. I hypothesize that there are extremely low triggering parameters of alternating electromagnetic field (EMF) intensity, i.e., above the background magnetic field on earth but below the lowest AEMF and PEMF that have been investigated to date (above 0.07 mT and below 0.4 mT) that induce cellular response. Methods: Accordingly, human monolayer explant culture replica were exposed four times in 24-hour intervals to two minutes of 10 kHz AEMF or PEMF (10 Hz pulses at a basic 5 kHz frequency) with a maximal EMF intensity of 0.2 mT for both. Cell proliferation was estimated from microscopic cell counting and cell death by lactate dehydrogenase (LDH) specific activity in culture media (measured using a colorimetric method). The early marker of osteoblast maturation, cellular alkaline phosphatase (AP) specific activity, was measured using a colorimetric method (n=6 for all experiment conditions). Results: No difference was found in cell numbers in the culture samples exposed either to AEMF or PEMF and in the LDH’s specific activity in culture media in comparison to the unexposed controls (p&gt;0.05, for both). The cellular AP’s specific activity increased significantly only in cell cultures exposed to the 10 kHz AEMF (p=0.011). Conclusions: The triggering for human osteoblast activation for maturation by an extremely low AEMF (10 kHz) is at least 0.2 mT, which is distinct and below the previously found triggering range of a PEMF for proliferation induction. Therefore, application of these EMF parameters in a clinical setup by a separate finetuning of osteoblast proliferation and maturation might have a therapeutic value in enhancing damaged bone regeneration.

Combining total and differential somatic cell count to screen for mastitis

Combining total and differential somatic cell count to screen for mastitis

Stability of canine and feline cerebrospinal fluid samples regarding total cell count and cell populations stored in \u201cTransFix\xae/EDTA CSF sample storage tubes\u201d

BackgroundBecause of fast leucocyte degeneration in cerebrospinal fluid (CSF) laboratory examinations of CSF samples should be performed approximately within 30 min after withdrawal. This study examines the storage of canine and feline CSF samples in “TransFix®/EDTA CSF Sample Storage Tubes” (Cytomark, Buckingham, UK) for preventing leucocytes from degeneration, so that routine and flow cytometry examinations are feasible up to 3 days after sampling.ResultsAfter storage in TransFix® tubes, leukocytes could not be adequately stained with Türk’s solution and differentiating between erythrocytes and leukocytes was cumbersome. In addition, the cell morphology could not be sufficiently assessed on cytospin preparations because of shrunken leukocytes and indistinct cell nuclei. In contrast, by flow cytometry, a significantly higher cell count was measured over the entire study period in the samples stored in TransFix® tubes compared to the untreated samples. The antibodies (AB) against CD3, CD4 and CD21, against CD11b and against CD45 showed a good binding strength and thus enabled a good differentiation of cell populations. However, after storage in the TransFix® tubes, monocytes were no longer detectable using an AB against CD14.ConclusionBased on these results, “TransFix®/EDTA CSF Sample Storage Tubes” can be used for extended storage prior to flow cytometric analysis of lymphocytes and granulocytes in CSF samples but not for detecting monocytes. However, standard examinations, such as microscopic cell counting and morphological cell assessment should be performed on fresh CSF samples.

Comparison of Cariogenic Characteristics between Fluoride-sensitive and Fluoride-resistant Streptococcus mutans

The aim of this study is to compare cariogenic characteristics of fluoride-sensitive &lt;i&gt;Streptococcus mutans&lt;/i&gt; [fluoride-sensitive (FS) &lt;i&gt;S. mutans&lt;/i&gt; ] and fluoride-resistant &lt;i&gt;Streptococcus mutans&lt;/i&gt; [fluoride-resistant (FR) &lt;i&gt;S. mutans&lt;/i&gt;] in the presence of sucrose, and to evaluate its effect on cariogenic biofilm formation. &lt;i&gt;S. mutans&lt;/i&gt; ATCC 25175 was continuously cultured in trypticase soy broth (TSB) containing NaF (70 ppm) for 40 days to generate FR &lt;i&gt;S. mutans&lt;/i&gt; . FS and FR &lt;i&gt;S. mutans&lt;/i&gt; were inoculated in TSB with or without 2% sucrose, and optical density and pH were measured every hour. An oral biofilm was formed using saliva bacteria and analyzed through confocal laser scanning microscopy and CFU count. Finally, the expression of glucosyltransferases genes of both &lt;i&gt;S. mutans&lt;/i&gt; was investigated through RT-PCR. FR &lt;i&gt;S. mutans&lt;/i&gt; exhibited slower growth and lower acidogenicity in the presence of sucrose compared to FS &lt;i&gt;S. mutans&lt;/i&gt; . Both cariogenic and single species biofilm formation was lower in the presence of FR &lt;i&gt;S. mutans&lt;/i&gt; , along with reduced number of bacteria. FR &lt;i&gt;S. mutans&lt;/i&gt; showed significantly low levels of gtfB, gtfC, and gtfD expression compared to FS &lt;i&gt;S. mutans&lt;/i&gt; . On the basis of results, FR &lt;i&gt;S. mutans&lt;/i&gt; may be less virulent in the induction of dental caries.

Prevalence, characteristics and risk factors of imported and local malaria cases in North-Western Province, Zambia: a cross-sectional study

BackgroundImported malaria is a major challenge for countries that are in malaria elimination stage such as Zambia. Legitimate cross-border activities add to the risk of transmission, necessitating determination of prevalence, characteristics and risk factors of imported and local malaria.MethodsThis cross-sectional study was conducted in 103 consented child and adult patients with clinical malaria symptoms, from selected health facilities in north-western Zambia. Patient demographic data and blood samples for malaria microscopy and full blood count were obtained. Chi-square and penalized logistic regression were performed to describe the characteristics and assess the risk factors of imported and local malaria in North-Western Province.ResultsOverall, malaria prevalence was 78.6% with 93.8% Plasmodium falciparum and 6.2% other species. The local cases were 72 (88.9%) while the imported were 9 (11.1%) out of the 81 positive participants. About 98.6% of the local cases were P. falciparum compared to 55.6% (χ2 = 52.4; p < 0.01) P. falciparum among the imported cases. Among the imported cases, 44% were species other than P. falciparum (χ2 = 48; p < 0.01) while among the local cases only 1.4% were. Gametocytes were present in 44% of the imported malaria cases and only in 2.8% of the local cases (χ2 = 48; p < 0.01). About 48.6% of local participants had severe anaemia compared to 33.3% of participants from the two neighbouring countries who had (χ2 = 4.9; p = 0.03). In the final model, only country of residence related positively to presence of species other than P. falciparum (OR = 39.0, CI [5.9, 445.9]; p < 0.01) and presence of gametocytes (OR = 23.1, CI [4.2, 161.6]; p < 0.01).ConclusionMalaria prevalence in North-Western Province is high, with P. falciparum as the predominant species although importation of Plasmodium ovale and Plasmodium malariae is happening as well. Country of residence of patients is a major risk factor for malaria species and gametocyte presence. The need for enhanced malaria control with specific focus on border controls to detect and treat, for specific diagnosis and treatment according to species obtaining, for further research in the role of species and gametocytaemia in imported malaria, cannot be overemphasized.

Machine Learning (ML) Can Successfully Support Microscopic Differential Counts of Peripheral Blood Smears in a High Throughput Hematology Laboratory

Background: Cytomorphology is the gold standard for quick assessment of peripheral blood and bone marrow samples in hematological neoplasms. It is a broadly-accepted method for orchestrating more specific diagnostics including immunophenotyping or genetics. Inter-/intra-observer-reproducibility of single cell classification is only 75 to 90%. Only a limited number of cells (100 - 500 cells/smear) is read in a time-consuming procedure. Machine learning (ML) is more reliable where human skills are limited, i.e. in handling large amounts of data or images. We here tested ML to differentiate peripheral blood leukocytes in a high throughput hematology laboratory. Aim: To establish an ML-based cell classifier capable of identifying healthy and pathologic cells in digitalized peripheral blood smear scans at an accuracy competitive with or outperforming human expert level. Methods: We selected &amp;gt;2,600 smears out of our unique archive of &amp;gt; 250,000 peripheral blood smears from hematological neoplasms. Depending on quality, we scanned up to 1,000 single cell images per smear. For image acquisition, a Metafer Scanning System (Zeiss Axio Imager.Z2 microscope, automatic slide feeder and automatic oiling device) from MetaSystems (Altlussheim, GER) was used. Areas of interest were defined by pre-scan in 10x magnification followed by high resolution scan in 40x to generate cell images for analysis. Average capture times for 300/500 cells were 3:43/4:37 min We set up a supervised ML-learning model using colour images (144x144 pixels) as input, outputting predicted probabilities of 21 predefined classes. We used ImageNet-pretrained Xception as our base model. We trained, evaluated and deployed the model using Amazon SageMaker on a subset of 82,974 images randomly selected from 514,183 cells captured and labelled for this study. 20 different cell types and one garbage class were classified. We included cell type categories referring to the critical importance of detecting rare leukemia subtypes (e.g. APL). Numbers of images from respective 21 classes ranged from 1,830 to 14,909 (median: 2,945). Minority classes were up-sampledto handle imbalances. Each picture was labelled by highly skilled technicians (median years practicing in this laboratory: 5) and two independent hematologists (median years at microscope: 20). Results: On a separate test set of 8,297 cells, our classifier was able to predict any of the five cell types occurring in the peripheral blood of healthy individuals (PMN, lymphocytes, monocytes, eosinophils, basophils) at very high median accuracy (97.0%) Median prediction accuracy of 15 rare or pathological cell types was 91.3%. For six critical pathological cell forms (myeloblasts, atypical/bilobulated promyelocytes in APL/APLv, hairy cells, lymphoma cells,plasma cells), median accuracy was 93.4% (sensitivity 93.8%). We saw a very high "T98 accuracy" for these cell types (98.5%) which is the accuracy of cell type predictions with prediction probability &amp;gt;0.98 (achieved in 2231/2417 cases), implicating that critical cells predicted with probability &amp;lt;0.98 should be flagged for human expert validation with priority. For all 21 classes median accuracy was 91.7%. Accuracy was lower for cells representing consecutive steps of maturation, e.g. promyelo-/myelo-/metamyelocytes, reproducing inconsistencies from the human-built phenotypic classification system (s.Fig.). Conclusions: We demonstrate an automated workflow using automatic microscopic cell capturing and ML-driven cell differentiation in samples of hematologic patients. Reproducibility, accuracy, sensitivity and specificity are above 90%, for many cell types above 98%. By flagging suspicious cells for humanvalidation, this tool can support even experienced hematology professionals, especially in detecting rare cell types. Given an appropriate scanning speed, it clearly outperforms human investigators in terms of examination time and number of differentiated cells. An ML-based intelligence can make its skills accessible to hematology laboratories on site or after upload of scanned cell images, independent of time/location. A cloud-based infrastructure is available. A prospective head to head challenge between ML-based classifier and human experts comparing sensitivity and accuracy for detection of all cell classes in peripheral blood will be tested to proof suitability for routine use (NCT 4466059). Figure Disclosures Heo: AWS: Current Employment. Wetton:AWS: Current Employment. Drescher:MetaSystems: Current Employment. Hänselmann:MetaSystems: Current Employment. Lörch:MetaSystems: Current equity holder in private company.

Surface properties and Streptococcus mutans - Streptococcus sanguinis adhesion of fluorotic enamel

ObjectivesThe aim of this in vitro study was to evaluate the surface properties of moderately to severely fluorotic enamel and the adhesion of Streptococcus mutans and Streptococcus sanguinis to enamel, exploring the relationship between dental fluorosis and dental caries from a microbiology perspective. MethodsWe examined the basic surface properties of moderately to severely fluorotic enamel by surface microhardness test, scanning electron microscopy (SEM) and atomic force microscopy. Then S. mutans single-species biofilms and S. mutans - S. sanguinis dual-species biofilms were cultured on fluorotic enamel surface. The morphology of biofilms, the volume of bacteria and expolysaccharides (EPS) and the number of bacteria were respectively tested by SEM, confocal laser scanning microscopy and colony-forming units (CFU) counting. ResultsFluorotic enamel displayed lower average microhardness and greater surface roughness than sound enamel, and it also showed structure defects like pores or pits. The biofilm thickness, volume of bacteria and EPS, and CFU counts of bacteria in both single-species and dual-species biofilms on fluorotic enamel were all significantly higher than those on sound enamel. The volume of bacteria and EPS in dual-species biofilms are both less than those of single-species biofilms. ConclusionsThe higher surface roughness and the structure defects of teeth with moderate to severe dental fluorosis contributed to the adhesion of S. mutans and S. sanguinis, and the increased adhesion of S. mutans may increase the susceptibility of dental caries. However, S. sanguinis would play a role as a “designer bacteria” which reduce the cariogenicity of the biofilms on fluorotic enamel surface.

Changes in heterotrophic microbial communities induced by biocidal treatments in the Monastery of San Martiño Pinario (Santiago de Compostela, NW Spain)

Previous studies have provided evidence that chemical biocides can condition the behaviour of microbial populations, thereby promoting recolonization by some groups of microorganisms. We conducted a year-long study to evaluate the extent to which fungal and bacterial communities are affected by the biocidal treatments Biotin R®, Biotin T®, Preventol RI80®, New Des 50®, benzalkonium chloride and an ethanol-water (1:1) mixture, used to remove Apatococcus lobatus from the granite walls of the San Martiño Pinario Monastery in Santiago de Compostela (NW Spain). Bacterial, fungal, algal and cyanobacterial concentrations were enumerated via microscopic counts of colony-forming units on agar plates, one, six and twelve months after application of the biocidal treatments. Cultivable species were further characterized by morphological taxonomic analysis (algae and fungi) and DNA analysis by sequencing of 16S rDNA gene (bacteria) and the ITS region plus the D1/D2 region of the 28S rDNA gene (fungi) before and after one-year treatment. None of the treatments led to overgrowth of bacteria or fungi, while benzalkonium chloride and water (used as control) treatments were the most effective in preventing bacterial recolonization and Biotin T® was the most effective in preventing fungal recolonization. Although the bacterial population did not increase in numbers after any of the treatments, the diversity was altered, with Bacillus halotolerans disappearing and Bacillus pumilus and Brachybacterium paraconglomeratum appearing in the areas treated with biocides. In fungi, Eutypa sp. was eliminated with all biocides except with Preventol RI80® and no species was promoted.

Using ITS2 metabarcoding and microscopy to analyse shifts in pollen diets of honey bees and bumble bees along a mass‐flowering crop gradient

Worldwide pollinator declines lead to pollination deficits in crops and wild plants, and managed bees are frequently used to meet the increasing demand for pollination. However, their foraging can be affected by flower availability and colony size. We investigated how mass-flowering oilseed rape (OSR) can influence the pollen resource use of small and large honey bee (Apis mellifera L.) and bumble bee (Bombus terrestris L.) colonies. Colonies were placed adjacent to strawberry fields along a gradient of OSR availability in the landscapes. We used ITS2 metabarcoding to identify the pollen richness based on ITS2 amplicon sequencing and microscopy for quantification of target pollen. Bumble bees collected pollen from more different plant genera than honey bees. In both species, strawberry pollen collection decreased with high OSR availability but was facilitated by increasing strawberry flower cover. Colony size had no effect. The relationship between next-generation sequencing-generated ITS2 amplicon reads and microscopic pollen counts was positive but pollen type-specific. Bumble bees and, to a lesser degree, honey bees collected pollen from a wide variety of plants. Therefore, in order to support pollinators and associated pollination services, future conservation schemes should sustain and promote pollen plant richness in agricultural landscapes. Both bee species responded to the availability of flower resources in the landscape. Although honey bees collected slightly more strawberry pollen than bumble bees, both can be considered as crop pollinators. Metabarcoding could provide similar quantitative information to microscopy, taking into account the pollen types, but there remains high potential to improve the methodological weaknesses.

The microbial community, its biochemical potential, and the antimicrobial resistance of Enterococcus spp. in Arctic lakes under natural and anthropogenic impact (West Spitsbergen)

The sustainable management of small human communities in the Arctic is challenging. In this study, both a water supply system (Lake 1) under the natural impact of a bird-nesting area, and a wastewater receiver (Lake 2) were analysed in the vicinity of the Polish Polar Station on West Spitsbergen. Microbial community composition, abundance and activity were assessed in samples of the treated wastewater, lake water and sediments using next-generation sequencing and direct microscope counts. Special attention was given to the faecal indicator, Enterococcus spp., whose occurrence and antimicrobial resistance were tested in water and wastewater samples. The results indicate that Lake 1, at a tundra stream discharge (L-TS) and a water supply point (L-WS) were dominated by three phyla: Proteobacteria (57–58%) Bacteroidetes (27–29%) and Actinobacteria (9–10%), showing similar microbial composition up to the genus level. This suggests that nutrient-rich runoff from the bird colony was retained by surrounding tundra vegetation and reached Lake 1 at L-TS to a limited extent. Lake 2, being the wastewater recipient (WW-R), mirrors to some extent the core phyla of treated wastewater (WW-E), but in different shares. This suggests the possible washout of wastewater-related bacteria with activated sludge flocs, which was also supported by the microscopic observations. Compared to Lake 1, in WW-R an increase in all tested parameters was noted: total prokaryotic cell number, average cell volume, prokaryotic biomass and live cell percentage. The presence of Enterococcus spp. antibiotic resistance patterns highlight the importance of human associated microbiome and resistome dissemination via wastewater discharge. Moreover, it can be expected that temperature-related biochemical processes (e.g. nutrient cycling) may be accelerated by the ongoing climate change. Thus, proper wastewater treatment requires locally adapted solutions in increasingly visited and inhabited polar regions. Additionally, microbial community discharged to the environment with the treated wastewater, requires critical attention.

Relationships of Alizarin Test Scores with Somatic Cell Count in Bovine Milk

The aim of this study was to reveal the association of alizarin test scores (ATS) with somatic cell scores (SCC) in bovine milk. The milk samples (n=60) were collected from three milk selling points in Samsun province of Turkey in two test days (TD). To obtain ATS, change of colour in milk after adding dye solution were used (ATS 1: red-brown/normal, ATS 2: yellow/acidic, and ATS 3: violet/alkali). To test for reliability of ATS by acidity, pH values of the samples were measured with a standard pH meter. In SCC analyses, direct microscopic cell counting was applied. Of examined parameters, only SCC was differed by TD, and both pH and SCC were differed by the location. According to ATP groups, pH and SCC means were also changed. Finally, estimated weak (r=0.247) correlation coefficient between ATS and SCC pointed out that ATS data are not suitable to detect quality degree of bovine milk samples.

Performance of the Kato-Katz method and real time polymerase chain reaction for the diagnosis of soil-transmitted helminthiasis in the framework of a randomised controlled trial: treatment efficacy and day-to-day variation

BackgroundAccurate, scalable and sensitive diagnostic tools are crucial in determining prevalence of soil-transmitted helminths (STH), assessing infection intensities and monitoring treatment efficacy. However, assessments on treatment efficacy comparing traditional microscopic to newly emerging molecular approaches such as quantitative Polymerase Chain Reaction (qPCR) are scarce and hampered partly by lack of an established diagnostic gold standard.MethodsWe compared the performance of the copromicroscopic Kato-Katz method to qPCR in the framework of a randomized controlled trial on Pemba Island, Tanzania, evaluating treatment efficacy based on cure rates of albendazole monotherapy versus ivermectin-albendazole against Trichuris trichiura and concomitant STH infections. Day-to-day variability of both diagnostic methods was assessed to elucidate reproducibility of test results by analysing two stool samples before and two stool samples after treatment of 160 T. trichiura Kato-Katz positive participants, partially co-infected with Ascaris lumbricoides and hookworm, per treatment arm (n = 320). As negative controls, two faecal samples of 180 Kato-Katz helminth negative participants were analysed.ResultsFair to moderate correlation between microscopic egg count and DNA copy number for the different STH species was observed at baseline and follow-up. Results indicated higher sensitivity of qPCR for all three STH species across all time points; however, we found lower test result reproducibility compared to Kato-Katz. When assessed with two samples from consecutive days by qPCR, cure rates were significantly lower for T. trichiura (23.2 vs 46.8%), A. lumbricoides (75.3 vs 100%) and hookworm (52.4 vs 78.3%) in the ivermectin-albendazole treatment arm, when compared to Kato-Katz.ConclusionsqPCR diagnosis showed lower reproducibility of test results compared to Kato-Katz, hence multiple samples per participant should be analysed to achieve a reliable diagnosis of STH infection. Our study confirms that cure rates are overestimated using Kato-Katz alone. Our findings emphasize that standardized and accurate molecular diagnostic tools are urgently needed for future monitoring within STH control and/or elimination programmes.

Monitoring of Peronospora destructor Primary and Secondary Inoculum by Real-Time qPCR.

Onion downy mildew (ODM), caused by Peronospora destructor, is a serious threat for onion growers worldwide. In southwestern Québec, Canada, a steady increase in occurrence of ODM has been observed since the mid-2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which act as initial inoculum locally and also for neighboring areas. It also produces oospores capable of surviving in soils and tissues for a prolonged period of time. A recent study showed that ODM epidemics are strongly associated with weather conditions related to production and survival of overwintering inoculum, stressing the need to understand the role of primary (initial) and secondary inoculum. However, P. destructor is an obligate biotrophic pathogen, which complicates the study of inoculum sources. This study aimed at developing a molecular assay specific to P. destructor, allowing its quantification in environmental samples. In this study, a reliable and sensitive hydrolysis probe-based assay multiplexed with an internal control was developed on the internal transcribed spacer (ITS) region to quantify soil- and airborne inoculum of P. destructor. The assay specificity was tested against 17 isolates of P. destructor obtained from different locations worldwide, other members of the order Peronosporales, and various onion pathogens. Validation with artificially inoculated soil and air samples suggested a sensitivity of less than 10 sporangia g-1 of dry soil and 1 sporangium m-3 of air. Validation with environmental air samples shows a linear relationship between microscopic and real-time quantitative PCR counts. In naturally infested soils, inoculum ranged from 0 to 162 sporangia equivalent g-1 of dry soil, which supported the hypothesis of overwintering under northern climates. This assay will be useful for primary and secondary inoculum monitoring to help characterize ODM epidemiology and could be used for daily tactical and short-term strategic decision-making.

Microbiological investigations of two thermal baths in Budapest, Hungary. Report: effect of bathing and pool operation type on water quality.

In Hungary, which is famous for its thermal baths, according to the regulations, waters are investigated in hygienic aspects with standard cultivation methods. In the present study, two thermal baths were investigated (the well and three different pool waters in both) using cultivation methods, taxon-specific polymerase chain reactions (PCRs), multiplex PCRs and next-generation amplicon sequencing. Mainly members of the natural microbial community of the well waters and bacteria originating from the environment were detected but several opportunistic pathogenic taxa, e.g., Pseudomonas aeruginosa, P. stutzeri, Acinetobacter johnsoni, Acinetobacter baumanni, Moraxella osloensis, Microbacterium paraoxydans, Legionella spp., Stenotrophomonas maltophilia and Staphylococcus aureus were revealed by the applied methods. Pools with charging-unloading operation had higher microscopic cell counts, colony-forming unit (CFU) counts, number of cocci, P. aeruginosa and S. aureus compared to the recirculation systems. Bacteria originating from human sources (e.g., skin) were identified in the pool waters with less than 1% relative abundance, and their presence was sporadic in the pools. Comparing the microbiological quality of the pools based on the first sampling time and the following four months' period it was revealed that recirculation operation type has better water quality than the charging-unloading pool operation from a hygienic point of view.

The new microbiology: cultivating the future of microbiome-directed medicine.

The discovery of human-associated microscopic life forms has captivated the scientific community since their first documentation in the 17th century. Subsequent isolation and cultivation of microorganisms have spurred great leaps in medicine, including the discovery of antibiotics, identifying pathogens that cause infectious diseases, and vaccine development. The realization that there is a vast discrepancy between the number of microscopic cell counts and how many could thrive in the laboratory motivated the advent of sequencing-based approaches to characterize the uncultured fraction of the microbiota, leading to an unprecedented view into their composition and putative function on all bodily surfaces. It soon became apparent that specific members of the microbiota can be our commensal partners with new implications on various aspects of health, as well as a rich source of therapeutic compounds and tools for biotechnology. Harnessing the immense repertoire of microbial properties, however, inadvertently requires pure cultures for validation and manipulation of candidate genes, proteins, or metabolic pathways, just as mammalian cell culture has become an indispensable tool for mechanistic understanding of host biology. Yet, this renewed interest in growing microorganisms, individually or as a consortium, is stalled by the laborious nature of conventional cultivation methods. Addressing this unmet need through implementation of improved media design and new cultivation techniques is arguably instrumental to future milestones in translational microbiome research.