Automatic Pipette Research Articles

Effects of Curcumin and Doxorubicin on the Viability of Neuroblastoma Cancer Cell Line

Aim: Neuroblastoma has a very important place among childhood diseases, and despite all the methods used in treatment, it is very difficult to prevent neuroblastoma invasion. The number of studies showing that curcumin, the most active component of turmeric, is not toxic, is increasing day by day. In this study, the anticancer activities of curcumin (Cur), one of the important active compounds, were demonstrated in the Neuroblastoma cancer cell line (Na2B). Method: Neuroblastoma cell line was used in the study. To determine the IC50 doses of Dox and Cur, Na2B cells line were cultivated with an automatic pipette. MTT analysis was performed to analyze cell survival (viability). Inhibition levels in the cells were determined at 24 and 48 hours. Results: While the IC50 of Na2B cell proliferation was approximately 124.5 uM at the 48th hour in Dox-treated cells, the IC50 value of Cur at the 48th hour was found to be 224.6 uM. Conclusion: These results showed that Cur could be an alternative agent in the treatment of neuroblastoma, and its fewer side effects compared to other chemotherapeutic agents such as Dox would increase its preferability.

Dependence of element composition of bile of freshwater and marine fishes on some abiotic and biotic factors

IntroductionThe elemental composition and physical properties of fish bile is a very important marker for ecotoxicological and physiological studies as bile is able to accumulate elements from environmental compartments. In the present study we focused on the effects of long term (seasons and years) and spatial distance (different water bodies) as well as other biotic (feeding habits, fish species, gut morphology) and abiotic (water salinity) factors on element composition of bile of 429 individuals belonging to 22 forms/species of fishes.MethodsElement composition was determined using an inductively coupled plasma optical emission spectrometry. The fishes were analyzed from one marine (the Kandalaksha Gulf of the White Sea), two freshwater oligotrophic (Teletskoye and Baunt lakes), and one freshwater eutrophic water body (Chany Lake). In parallel, we have estimated the volume and density of bile from the same fishes using an automatic pipette and electronic balance.Results and discussionBased on fish bile density and volume data, we hypothesize that gastric fish species (those fishes that have a stomach) require a low volume of bile, but with higher density if compared to agastric fish species (those lacking a stomach) that mainly possessed large volumes of bile, but with a lower density. We have found that the concentrations of the following Na, K, Ca, Mg, S, P (major elements) and Al, Cu, Fe, Sr, Zn (trace elements) were the highest among the 28 elements studied. There were significant findings to contribute to a better understanding of the physiology of bile. First, we have determined the conditions formed in a water body in a given year (season) are more important for element composition of fish bile rather than other tested factors (feeding habits, gut morphology, etc.). Second, the feeding habits of fishes (carnivorous compared to non-carnivorous) had a significant effect based on differences among several elements. Third, the clear distinction between marine and freshwater fishes was found only for K and Na. Understanding which elements are produced as part of waste elimination and those that are present as a consequence of vital biological functions could improve the ecotoxicological study of bile as a marker of heavy metal contamination.

A rapid and convenient enzyme digestion method for the analysis of N-glycans using exoglycosidase-impregnated polyacrylamide gels fabricated in an automatic pipette tip.

Efficient enzymatic digestion methods are critical for the characterization and identification of glycans. Glycan hydrolysis enzymes are widely utilized for the identification of glycoprotein or glycolipid glycans. The commonly utilized in solution glycan hydrolysis methods require several hours of incubation with enzymes for complete removal of their target monosaccharides. To develop an efficient and simple method for the rapid release of monosaccharides from glycoprotein glycans, we fabricated exoglycosidase-impregnated acrylamide gels in an automatic pipette tip. Our automated enzymatic reactors are based on the simple photochemical copolymerization of monomers comprising acrylamide and methylene-bis-acrylamide-containing enzymes with an azobis compound functioning as the photocatalytic initiator. After filling the tip of the automatic pipette with these acrylamide solutions, polymerization of the acrylamide gel solution was performed by irradiation with a LED. The immobilized enzymes maintained their activities in the pipette tips and their action was completed by fully automatic pipetting for 10 to 30min. We utilized 8-aminopyrene-1, 3, 6-trisulfonic acid (APTS)-labeled glycans as a substrate and measured by capillary electrophoresis (CE) before and after enzymatic digestion. We demonstrated that this method exhibited quantitative enzymatic and specific cleavage of monosaccharides from glycoprotein glycans.

Analysis of microsamples by miniaturized magnetic-based pipette tip microextraction: determination of free cortisol in serum and urine from very low birth weight preterm newborns.

Miniaturized magnetic-based pipette tip microextraction is presented as a sample preparation approach for microsamples. It involves quick dispersion of a diminutive amount of a magnetic sorbent material in a low-volume sample (10 μL) to entrap the target analytes. Next, the dispersion is aspirated using a (semi)automatic pipette through a pipette tip with a small cubic neodymium magnet inside, which retrieves the magnetic sorbent containing the analytes. After discarding the rest of the sample, the sorbent is properly rinsed by aspirating/dispensing deionized water, and then, the analytes are eluted by aspirating/dispensing an appropriate solvent. This approach was employed for the determination of free cortisol in serum and urine from very low birth weight preterm newborns, a vulnerable patient group who present low availability for sampling biological fluids. A magnetic immunosorbent made of a cortisol antibody was employed for the selective extraction, followed by liquid chromatography-tandem mass spectrometry. Good analytical features were obtained, such as limits of detection and quantification of 0.08 and 0.27 ng mL-1, respectively, linearity up to 50 ng mL-1 (R2 > 0.999), RSD values under 15% and relative recoveries between 91 and 111%. The cross-reactivity with other glucocorticoids (i.e., cortisone and prednisolone) was evaluated to show the selectivity of the extraction. Finally, the method applicability was demonstrated towards the determination of free cortisol in the serum and urine samples from low birth weight preterm newborns.

Compatibility of fungicides with Trichoderma asperelloides and Azospirillum brasilense

The use of fungicides to control agricultural diseases has been increasing in agriculture. Thus, the biological control using fungus is an effective tool in the integrated diseases management. This study aimed to evaluate the compatibility of fungicides Certeza, Apron and Vitavax on the microorganisms Trichoderma asperelloides and Azospirillum brasilense. The experiments were conducted in vitro in a completely randomized design with a factorial arrangement 2 x 3 with three replications. The first factor composed of two doses and the second referred to the fungicides. The microorganisms were developed in PDA medium. After the medium has solidified, in a flow layer 10 µL of the fungal solution was added to each plate with the aid of an automatic pipette (D1, recommended dose; D2, double of recommended dose). Then, each Petri plate was centrally inoculated with a disc of Trichoderma asperelloides, in addition to the uninoculated control. For Azospirillum brasilense, the colony surface was scraped with the aid of a sterilized Drigalsky loop. Then, the filter of discs paper was dipped in the fungicides solution and placed on the medium surface. After this process, the plates were incubated in a BOD (Biochemical Oxygen Demand) at 26±2ºC and photoperiod 12 h. The parameters evaluated were a mycelial growth and sporulation. The results showed that there was no significant interaction between the factors. The fungicides Certeza (Tiofanato metílico + Fluazinam), Apron (Fludioxonil + Metalaxil-M) and Vitavax (Carboxina + Tiram) are considered compatible with Trichoderma asperelloides and Azospirillum brasilense. The fungicides can be used in an integrated disease management practices, priority must be given to use the products that have been shown to be less harmful.

A Real-Life Reproducibility Assessment for NMR Metabolomics.

Nuclear magnetic resonance (NMR) metabolomics is currently popular enough to attract both specialized and non-specialized NMR groups involving both analytical trained personnel and newcomers, including undergraduate students. Recent interlaboratory studies performed by established NMR metabolomics groups demonstrated high reproducibility of the state-of-the-art NMR equipment and SOPs. There is, however, no assessment of NMR reproducibility when mixing both analytical experts and newcomers. An interlaboratory assessment of NMR quantitation reproducibility was performed using two NMR instruments belonging to different laboratories and involving several operators with different backgrounds and metabolomics expertise for the purpose of assessing the limiting factors for data reproducibility in a multipurpose NMR environment. The variability induced by the operator, automatic pipettes, NMR tubes and NMR instruments was evaluated in order to assess the limiting factors for quantitation reproducibility. The results estimated the expected reproducibility data in a real-life multipurpose NMR laboratory to a maximum 4% variability, demonstrating that the current NMR equipment and SOPs may compensate some of the operator-induced variability.

Investigation of the effects of different disinfectant solutions on honey bees (Apis mellifera )

In this study, it was aimed to investigate the toxic effects of biocidal and nano silver-containing disinfectants, which were used in beekeeping, on bees. Biocidal and nano-silver-containing preparations used in disinfection of hives were obtained from commercial companies. Syrup (1/1 sucrose-water) was given to the control group (Group 1; n = 10). Biocidal preparation (Group 2; n = 10) and nano-silver containing preparation (Group 3; n = 10) were given to one of the experimental groups via an automatic pipette, orally 2 µl per bee. 24 hours after the application, the bees that died in all groups were counted and the midgut tissues of the bees that survived in the groups were taken for histomorphological analysis. No application was performed in the control group (Group 1). Different disinfection solution was used in the group 2 (biocidal ingredient) and Group 3 (nano silver contents). The preparations were applied to the groups by spraying and bee deaths were recorded. Two disinfectants applied to the hives under field conditions, were found to cause more bee deaths than the control group. The highest bee death was in the nano silver group. In laboratory trials, the nano-silver-containing preparation was observed to cause high number of bee deaths and serious damage to the midgut epithelium in histomorphological examinations. The results of the study showed that direct application of disinfectant substances on bees caused serious deaths in the colony. Biocidal and chemical based preparations and hive disinfection should be applied in the empty beehives.

Analysis of Salivary Metabolites by Nuclear Magnetic Resonance Before and After Oral Mucosa Cleaning of Infants in the Pre-dental Period

The aim of the present study was to verify if a protocol for cleaning the oral cavity of infants in the pre-dental period can reduce extrinsic salivary metabolites observed through Nuclear Magnetic Resonance (NMR). A cross-sectional clinical study with a convenience sample was conducted, and infants were recruited at the UFRJ Pediatric Dentistry Clinic. Participants who had used antibiotics and/or antifungals up to 3 months before and whose legal guardians did not consent or sign the Informed Consent Form were excluded. An anamnesis was performed with the guardians and the participants' intraoral clinical examination. Initial collection of unstimulated total saliva was performed using an automatic pipette with sterile plastic tips in the buccal floor region, at least 1 h after the last feeding. Subsequently, the infants' oral mucosa was cleaned with gauze moistened with filtered water, and after 5 min, a new collection was performed, using the same methodology. The obtained samples were immediately transferred on ice to the laboratory, centrifuged (10,000 g), and stored at −80°C. The NMR analyses were performed using a 500-MHz spectrometer Bruker, Germany); evaluations were done via the 1H and 1H-1H TOCSY spectra for metabolite signaling. Eleven pre-dental infants were evaluated, with a mean age of 3.8 months, including six girls (55%). Of these, nine participants (82%) were exclusively breastfed. The higher presence of components such as lactose, glucose, sugars, acetate, alanine, and lactate were observed in the samples before oral mucosa cleaning. Regarding the type of diet, more lactose was observed in the saliva of patients who were exclusively breastfed than those that received mixed feeding. We conclude that the oral mucosa cleaning of infants in the pre-dental period tends to reduce the concentration of extrinsic components from the diet, such as lactose, in the salivary metabolomic profile analyzed by NMR.

Chemosensitive Thin Films Active to Ammonia Vapours.

The paper presents various dispersive systems developed for sensing toxic substance—ammonia. Polycarbonate dissolved in methylene chloride was used as a polymer matrix, which was enriched with: multi-walled carbon nanotubes (MWCNs), reduced graphene oxide (rGO) and conductive polymer (polyaniline—PANi). Dispersive systems were applied to the prefabricated substrates with comb electrodes by two methods: spraying and drop-casting, forming an active chemosensitive to ammonia vapours films. The spraying method involved applying the dispersion to the substrate by an aerograph for a specific time, whereas drop-casting involves depositing of the produced dispersive systems using a precision automatic pipette. The electrical responses of the obtained films were examined for nominal concentrations of ammonia vapours. Different types of dispersions with various composition were tested, the relationships between individual compounds and ammonia were analysed and the most promising dispersions were selected. Sensor containing rGO deposited by drop-casting revealed the highest change in the resistance (14.21%).

Herbicidal potential of Araucaria angustifolia (Bertoloni) Otto Kuntze in lettuce seedlings in vitro

Araucaria angustifolia (Bertoloni) Otto Kuntze plants are a viable source of potentially active secondary metabolites; however, deep studies and researches about the activity of these compounds are needed. The objective of this work was to assess the herbicidal activity of ethanolic extracts of plants of the species A. angustifolia in lettuce seedlings in vitro. Ethanolic extracts at different concentrations (0,0%, 12.5%, 25.0%, 50.0%, and 100.0%) were prepared using barks and leaves. A semisolid MS medium with pH adjusted to approximately 5.7 was prepared, solidified with 8g L-1 of agar, and autoclaved at 1.6atm for 20 minutes. The extracts were added to the culture medium during their preparation before the autoclaving, using an automatic pipette containing 0.1mL of A. angustifolia extract per lettuce seedling. The number of leaves per seedling, seedling height, chlorophyll content, root and shoot dry weights, and mortality percentage was determined at 20 days after inoculation. The results showed that the extract from leaves or barks of A. angustifolia plants has herbicidal activity in lettuce seedlings in vitro, and the extract concentration of 100% presented the best inhibitory results for the variables evaluated.

Refining drug administration in a murine model of acute infection with Trypanosoma cruzi

BackgroundIn animal research, “refinement” refers to modifications of husbandry or experimental procedures to enhance animal well-being and minimize or eliminate pain and distress. Evaluation of drug efficacy in mice models, such as those used to study Trypanosoma cruzi infection, require prolonged drug administration by the oral route (e.g. for 20 consecutive days). However, the orogastric gavage method can lead to significant discomfort, upper digestive or respiratory tract lesions, aspiration pneumonia and even accidental death. The aim of this work was to evaluate the effect of two administration methods (conventional oral gavage vs. a refined method using a disposable tip and automatic pipette) on the efficacy of benznidazole in a murine model of T. cruzi infection.ResultsBoth administration methods led to a rapid and persistent reduction in parasitaemia. Absence of T. cruzi DNA (evaluated by real-time PCR) in blood, cardiac and skeletal muscle confirmed that treatment efficacy was not influenced by the administration method used.ConclusionsThe proposed refined method for long-term oral drug administration may be a suitable strategy for assessing drug efficacy in mice models of Chagas disease and can be applied to similar murine infection models to reduce animal discomfort.

Development of an interactive simulation and app (Pipette Master) to teach undergraduate students how to read, set and use and automatic laboratory pipette

Abstract An automatic pipette is a key piece of laboratory equipment. The ability to accurately and reproducibly deliver any volume is a critical skill to master for all undergraduate students in the laboratory sciences. Over many years we have noted that students struggle in learning several facets of correct pipette usage. These include, choosing the correct pipette for a given volume, correctly setting the pipette and correctly using the plunger to aspirate and dispense the liquid. Students also struggle with unit conversions between ml and μl and vice versa. Lastly, students fail to retain these skills and information in following semesters of teaching. In an attempt to help students with converting between units (μl and ml) as well as choosing the most appropriate pipette for a given volume, I developed an interactive simulation which covered all these major aspects. Using a Gilson pipette as an example, the simulation covered how to set the pipette for a number of defined volumes. An Android/iOS app of the simulation has also been developed which can be used during laboratory classes. In 2017, we polled first year undergraduate students about their level of experience, knowledge of pipettes and level of comfort in converting between units and we then explained the simulation to them. The feedback on their skills and the software will be discussed. We also tested the impact of the simulation using 2nd year students enrolled in genetics. Using a laboratory quiz, we found significant improvements in student performance when students used the simulation compared to those that did not. We suggest this interactive approach is a simple and effective way to enhance student learning of key aspects of laboratory pipettes. The simulation is freely available.

Protective Effect of Apelin-13 on Kidney and Platelet Functions in Adenine-Induced Chronic Renal Failure in Rats

AbstractBackground: Apelin and angiotensin I receptor-related protein J receptor (APJ) were found to be expressed in the kidney. Also, in cases of Chronic Renal Failure (CRF), platelet function was found to be affected. Apelin-13 treatment effect on both kidney and platelet functions in cases of CRF was not fully understood.Aim of Study: To investigate the possible protective effect of apelin-13 on kidney and platelet function in adenine-induced CRF in rats.Material and Methods: Rats were divided into three groups of eight rats each as follow: Intact control group received a standard diet for 28 days, adenine treated group received a standard diet added to it adenine (0.75% w/w) for 28 days, and adenine + apelin treated group received a standard diet added to it adenine as in the adenine treated group with a concomitant treatment with apelin-13 for 28 days. Apelin-13 was dissolved in saline immediately before use and was given at a dose of 5μg/kg body weight/day subcutaneously. 24-hour urine samples were collected, and 24-hour water intake was estimated on the 28th day of the study, while each rat was kept in a separate metabolic cage. The collected urine samples were measured for volume, total protein, and creatinine levels. After 28 days from the start of the experimental period, rats were fasted overnight, and their blood pressure was measured using the Power Lab. Blood samples were collected and a part of it was placed in heparinized tubes to investigate platelet indices and another part of blood was kept in non-heparinized tubes which were left to clot for 30min at room temperature. Clotted blood was centrifuged at 3000rpm for 15min. The supernatant serum was pipetted off using fine tipped automatic pipettes and stored at –20oC until assayed.Results: Treatment with adenine (0.75%, w/w) for 28 days caused a significant decrease (p<0.001) in Body Mass Index (BMI) and a significant increase (p<0.001) of the relative kidney weight when compared with that of the control group. On concomitant treatment with apelin-13 and adenine significantly mitigated (p<0.001) the adenine-induced reduction in BMI and the increase in relative kidney weight. Water intake and urine output of adenine treated rats were signifi-cantly higher (p<0.001) than that in control rats, while, simultaneous treatment with adenine and apelin-13 signifi-cantly decreased (p<0.001) both in comparison to adenine treated group. Also, in comparison with control group, the serum levels of creatinine, uric acid and urea were enhanced significantly (p<0.001) in adenine treated group, indicating that adenine-induced CRF model has been successfully estab-lished in this experiment. However, these enhancements were significantly decreased (p<0.001) by concomitant treatment with apelin-13. On the other hand, there was a significant decrease (p<0.001) in the creatinine clearance in adenine treated group in comparison to the control one, however, simultaneous treatment with adenine and apelin-13 signifi-cantly deteriorated this effect. Also, in this study, comparing to those of normal group, the Superoxide Dismutase (SOD) and Glutathione Reductase (GR) levels in adenine treated group were significantly decreased (p<0.001) while, Malonal-dehyde (MDA) was significantly increased (p<0.001). The alteration of SOD, GR and MDA serum levels was remarkably reversed by concomitant treatment with apelin-13. A significant increase (p<0.001) in platelet count, Mean Platelet Volume (MPV), Platelet Distribution Width (PDW) and plateletcrit, but a significant decline (p<0.001) in bleeding time in adenine treated group in comparison to the control one. These changes were significantly reversed (p<0.001) by the concomitant treatment with adenine and apelin-13 in comparison to the adenine treated group.Conclusion: Apelin-13 treatment improved both kidney and platelet functions, and may give a promising strategy for slowing the progression of CRF and its complications.

High Throughput Experimental Methodologies for Accelerating Solid State Lithium-Ion Battery Development

Breakthrough in lithium-ion battery technology is crucial for modern sustainable energy applications, especially the electric vehicles. Compared to conventional lithium-ion battery with liquid electrolyte, solid state battery with solid inorganic electrolyte or flexible composite electrolyte possesses the advantages of higher energy density, higher working voltage, non-flammability, and lithium dendrite prevention [1, 2]. One of the main challenges of developing this next generation of batteries is to find the optimized structure and composition of solid electrolyte material, which usually has a ternary, quaternary, or even more complex composition. High throughput experimental methodologies, in which dozens of parallel experiments are carried out with small sample quantities per experiment, can greatly accelerate new material discovery and process optimization for solid electrolyte and solid state lithium ion battery development.To support this high-throughput experimental demand, the experimental instrument must meet requirements such as maximizing productivity through parallel experiments and automation, working with small amount of sample per experiment, and compatibility with Ar gas glove box operation. MTI Corporation (Richmond CA, USA) strive to provide highly efficient and economical experimental equipment and solutions for high throughput battery development. In this work, MTI Corporation¡¯s efforts on developing high throughput experimental solutions for solid state lithium-ion battery are discussed.The first step is solid inorganic electrolyte powder synthesis and pellet fabrication. For solid dispensing, four or more dispensing heads and balances, with one dispensing head for each solid electrolyte component, are integrated with a carousel type sample changer for automatic powder dispensing of 32 electrolyte powder samples. Next, the powder samples are ball milled in a planetary ball mill with 4 sets of 4 cavities milling jars for totally 16 parallel experiments. Then, the electrolyte powders are pressed into pellets for coin cell assembly using a hydraulic press with carousel type 16-sample changer. The powders and pellets are sintered, annealed, or quenched in a compact, 16-channel tube furnace up to 1700 ¡ãC with quenching option.Novel sintering techniques, such as spark plasma sintering (SPS) [3] and hydrothermal-assisted cold sintering process (CSP) [4], are employed for solid inorganic electrolyte sintering. These techniques, whether for the fast processing time of spark plasma sintering, or the low temperature processing capability of cold sintering process, open up possibilities of low cost, rapid fabrication of solid electrolytes, cathode pellets, or even the whole solid electrode-electrolyte stack [3].Solid inorganic electrolyte is usually too brittle for practical use. Composite electrolyte, which is made by embedding solid inorganic electrolyte powder in flexible polymer electrolyte, is a promising solution. By varying the ratio between inorganic and polymer components, a balance between electrical and mechanical properties can be achieved [4, 5]. The process of composite electrolyte fabrication starts with powder and liquid dispensing. For liquid dispensing, an automatic pipette robot with integrated XYZ stage is used for dispensing organic solvents and liquid electrolytes. The as-prepared high viscosity samples are mixed in a planetary centrifuge mixer for mixing 6 samples of 5 ml in one run. With addition of a 4-channel film applicator, existing doctor blade coater can be modified into high throughput equipment. The as-prepared sample with four strips of different composite electrolytes can be passed onto hot calendar machine or hot press for solvent evaporation and thickness reduction, and coin cell die cutter for high throughput preparation of composite electrolyte discs.Finally, the solid electrolyte powder and discs are analyzed by a compact X-ray fluorescence (XRF) spectrometer integrated with a XY sample stage for high throughput composition characterization. 32 samples can be qualitatively and quantitatively analyzed in one auto run. For high throughput assembling of coin cells, one route is to use an automatic coin cell crimper machine to facilitate coin cell assembling process. The other route is to use multiple split cells with quick, easy, repeatable assembling for parallel comparison of different solid electrolyte materials. This further saves time and labor by eliminating the coin cell crimping step.In conclusion, high throughput experimental solutions accelerate new material discovery and process optimization for solid state lithium ion battery research. Existing experimental techniques are modified for high throughput application by smart design of sample fixture, by integration, and by automation. Novel processing methods, such as spark plasma sintering and cold sintering, enable reduced processing time and low production cost.

Antibiograms in five pipetting steps: precise dilution assays in sub-microliter volumes with a conventional pipette.

We demonstrate a standalone microfluidic chip that allows us to carry out commonly executed antibiotic susceptibility assays in an array of nanoliter droplets. We eliminated the need for automation in performing an exemplary complicated liquid handling assay on a chip. Operations on droplets are hard-wired into the microfluidic chip. The liquid handling protocol can be executed with a simple and commonly available source of flow such as an automatic manual pipette. The system passively prepares a series of dilutions of a chemical compound and mixes them with portions of the sample. The precision of metering, merging, mixing, and splitting of discrete portions of liquid samples is rooted in the passive capillary action in microfluidic traps and not in the precision of dosing with a pipette. We show an exemplary use of the device in the determination of the minimum inhibitory concentration (MIC) of ampicillin against E. coli ATCC 25922.

Impact of Calibration of pipette on Quality Control Results

Background: Majority of biochemical investigations requires pipette for delivery of reagents and samples in clinical laboratory so that pipette that is used for the same must be properly calibrated for accurate results. Aims: The aim of this study to investigate that the impact of automatic pipette calibration on quality control results. Methodology: Total 6 pipettes are used in our study. Among them 3 pipettes are with volume 1.0 ml and another 3 are with volume 0.1 ml, all pipettes were examined for work calibration. Distilled water weighing method is used for calculation of accuracy and precision of pipettes. Control sera of glucose and uric acid is used for determining accuracy and Precision of pipettes. Result & Conclusion: Uncalibrated pipette had high effect on accuracy of control data as well as patients results therefore it is recommended that all pipette that is used in clinical laboratory must be periodically checked for calibration. Keywords: Accuracy, Calibration, Gujarat, Pipette, Precision

Detection of Hepatitis C virus in different samples with GeneXpert® HCV Viral Load kit

No: 1546 Presentation at ESCV 2015: Poster 2 Detection of Hepatitis C virus in different samples with GeneXpert® HCV Viral Load kit O. Janiekova1, J. Gasparovie1, H. Stigova2 1 National Reference Center for Viral Hepatitis, Bratislava, Slovakia 2 GTRADE s.r.o. Bratislava, Slovakia Background: Chronic HCV (CHC) infection is a global publichealthproblem.Recent studies estimate that 185millionpeople are currently infected worldwide and 3–4 million new infections are reported annually. ChronicHCV infection can cause severe liver disease including cirrhosis and hepatocellular carcinoma (HCC) with an interval of 20–30 years after being exposed to HCV. Molecular tests that detect and/or quantify HCV RNA are important in the diagnosis and management of patients with chronic Hepatitis C (CHC) undergoing anti-viral therapy. HCV RNA viral load monitoring is used to guide treatment duration where decisions can be made on-therapy and to determinewhether or not to stop therapy. GeneXpert® HCV Viral Load kit (Cepheid) can be used to measure HCV RNA levels at baseline and during treatment, to aid HCV diagnosis and to help predict sustained and no sustained virological responses to HCV therapy. Methods: We detected HCV RNA levels by using GeneXpert® HCV Viral Load cartridge in our 50 samples. Collected 5ml whole blood in a serum tubes were centrifuged and transferred 1ml of serum into Sample Chamber of cartridge via automatic pipette. Afterwards, GeneXpert® HCV Viral Load cartridge was loaded into GeneXpert® instrument and the analysis was launched. Based on the GeneXpert® technology, where RNA extraction is automated within the cartridge and GeneXpert® HCV Viral Load utilizes automated reverse transcriptase polymerase chain reaction (RT-PCR) using fluorescence to detect and quantify RNA. We obtained our results in 100min. Results: Samples in our studywere obtained from patients during and after treatment as well. We had one sample of healthcare person that was exposed to potential infectious source and one sample of sera from woman after childbirth. In our results 56 per cent of samples were negative, mainly, blood donors and patients after treatment. The remaining44per centof our sampleswerepositives.We detected the presence of viral RNA, especially in patients with primary diagnostics of Hepatitis C and patient with relapse of HCV infection. From whole number of positives samples, we obtained 35 percent high positive, with reliable results even from diluted samples. The range of viral load detected in our samples was from 450 IU/ml to 3, 84 million IU/ml. Conclusion: GeneXpert® HCV Viral Load kit is useful for the screening and early diagnosis of HCV infection. Thanks to high sensitivity and wide range of this test we detected HCV RNA levels in samples from different patients and in diluted samples too. http://dx.doi.org/10.1016/j.jcv.2015.07.204

First Report of a Garlic Bulb Rot Caused by Erwinia persicina in Europe

HomePlant DiseaseVol. 99, No. 5First Report of a Garlic Bulb Rot Caused by Erwinia persicina in Europe PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a Garlic Bulb Rot Caused by Erwinia persicina in EuropeL. Gálvez, J. Gil-Serna, M. García-Díaz, and D. PalmeroL. GálvezSearch for more papers by this author, J. Gil-SernaSearch for more papers by this author, M. García-DíazSearch for more papers by this author, and D. PalmeroSearch for more papers by this authorAffiliationsAuthors and Affiliations L. Gálvez J. Gil-Serna , Universidad Politécnica de Madrid (UPM), EUIT Agrícola, 28040 Madrid, Spain M. García-Díaz , Plant Genetic Resources Centre, INIA, 28800 Alcalá de Henares, Spain D. Palmero , Universidad Politécnica de Madrid (UPM), EUIT Agrícola, 28040 Madrid, Spain. Published Online:29 May 2015https://doi.org/10.1094/PDIS-11-14-1195-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In July 2013, symptoms of pinkish rot were observed in postharvest Chinese type garlic bulbs (Allium sativum L.) collected from Tembleque (Toledo, Spain). Fifty percent of the bulbs displayed symptoms of pink soft rot in at least one clove showing the pink color along all the flesh. Symptomatic cloves were surface disinfested for 3 min in a 2% sodium hypochlorite solution, rinsed twice with sterilized distilled water, and cultured for 2 days in trypticase soy agar (TSA). Gram-negative, oxidase negative, catalase positive, and facultatively anaerobic, motile, rod-shaped bacteria that were positive for glucose fermentation and nitrate reductase were consistently isolated from symptomatic tissues. Single colonies were light beige, circular, convex with entire margins, and pink-pigmented in PDA but not in TSA. Partial sequences of the 16S rRNA gene (1,386 bp) of two of the isolated bacterial strains were obtained using the primer pair p8FPL and p806R (McCabe et al. 1999) and deposited in EMBL database (accession nos. LM651373 and LM651372). BLAST analysis of the 16S rDNA sequences showed a 99% nucleotide sequence identity with the corresponding sequences of this gene previously available on the NCBI database of Erwinia aphidicola (accession no. NR_104724), E. persicina (NR_114078), and E. rhapontici (KF500098). Additionally, the gene encoding the RNA polymerase beta subunit was sequenced using primers ErpoBF (5′-GGACATGATCAACGCCAAGCCAA-3′) and ErpoBR (5′-GCACCCATCAATGCGCGGTTG-3′) designed in this work on the basis of different species of Erwinia genus. The sequences (581 bp, accession nos. KP100068 and KP100067) showed high identity (99%) with those of E. persicina previously available in databases (i.e. JF311799 and HQ393635) and 97% with the same gene in E. rhapontici (JF311793 and JF311794). The identification was completed with the sorbitol (positive result) and xylitol (negative) fermentation tests that confirmed the strains as E. persicina (Moretti et al. 2011). Pathogenicity tests were performed on Chinese garlic cloves using two E. persicina isolates. Ten cloves were disinfected in 5% sodium hypochlorite solution for 20 min and rinsed twice with sterile distilled water. Thereafter, cloves were wounded with a 200 µl pipette sterile tip (5 mm depth) and inoculated with 10 µl of bacterial suspension (adjusted to a concentration of 107 CFU/ml of TSB) using an automatic pipette. Subsequently, cloves were incubated in petri dishes with a paper towel that was soaked with sterilized distilled water. Ten control cloves were wounded and inoculated with sterilized TSB as negative controls. Inoculated garlic cloves were incubated at 28°C for 10 days in the dark. Pink rot symptoms were observed after 5 days on the inoculated cloves. Control cloves remained symptomless and no bacterium was reisolated. The experiment was repeated once obtaining the same results in both cases. All bacteria reisolated from the pink lesions were identified as E. persicina using the methodology described above. E. persicina has been previously isolated from lucerne, soybean, bean, and pea (González et al. 2007; Zhang and Nan 2014). To our knowledge, this is the first report of clove rot caused by E. persicina in garlic in Europe.

Do the values of prostate specific antigen obtained from fresh and dried urine reflect the serum measurements?

Aim:To investigate if free PSA (fPSA) and total PSA (tPSA) values obtained from simultaneously collected urine, fresh and dried on filter paper, reflect the serum free and total PSA.Materials and Methods:The sera and 20 cc first voided urine from 33 consecutive men aged between 40 and 84 (mean 61 ± 12), were collected in the morning and delivered to the laboratory. Three different aliquots of 100 microgram urine were taken with automatic pipette and dropped on 3 certain areas of a filter paper and allowed to dry for each patient. On each paper, borders of dried urine were marked. PSA values were obtained from the sera and fresh urine samples and recorded. Later on particular days dried urine samples were dissolved and eventually PSA values were derived and recorded again. The results were compared to each other. Correlations were evaluated by using an SPSS statistics program.Results:Serum PSA values correlated weakly (r < 0.24) with fresh and dried urine PSA values. While PSA in fresh and dried urine samples showed strong correlation (0.5 < r < 0.74), a very strong correlation (r > 0.75) among PSA values of dried urine samples of 1-day, 7- and 28-days, were seen.Conclusions:We conclude that PSA values obtained from fresh and dried urine could not reflect serum PSA values. But, because dried urine on a filter paper can be stable for years, it could be used for forensic purposes.

Fabrication of autoclavable bacteriologic loops for handling Mycobacterium tuberculosis isolates from recycled materials in a resource poor setting

In resource limited settings, the appropriation of scarce resources during research efforts can be daunting. Sourcing for disposable plastic bacteriological loops for manipulating M. tuberculosis had been eating into the research budget. In an attempt to reduce cost, an alternative and more cost effective way of obtaining autoclavable bacteriologic inoculation loops from used materials in the laboratory was employed. Autoclave resistant loops were prepared from polypropylene automatic pipette tips and platinum wires from electric stoves. The loop volume, when desired, was calculated using a simple mathematical equation after several passes in weighted water. Laboratories in resource poor settings could also save on inoculating loops by adopting such pragmatic approaches using recycled materials.