Optimal Dilution Research Articles

Optimized Culture Conditions for the Detection of Selected Strains of Bacillus in Eye Creams

Although eye area cosmetics contain preservatives, contamination can still occur during or after manufacture or through use. Understanding the likelihood of bacterial survival in eye creams begins with sensitive and accurate methods for the detection of bacterial contamination; therefore, we investigated optimal culture conditions, including neutralizers, dilution broths, and selective media for the detection of Bacillus in eye cream. Samples of three different brands of eye creams were first mixed with Tween 80, Tween 20, or a blend of Tween 60 and Span 80, then neutralized and non-neutralized samples were individually inoculated with B. cereus strains, B. mycoides, a mislabeled B. megaterium, B. subtilis or B. thuringiensis at a final concentration of 5 log CFU/g. The inoculated samples, with and without neutralizers, were spiral-plated and incubated at 30 °C for 24 h to 48 h. Presumptive colonies of Bacillus were enumerated on U. S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) referenced agars Bacillus cereus rapid agar (BACARA) and mannitol-egg yolk-polymixin agar (MYP). Our results show significant differences among the neutralizers, plates, and products. The combination of Tryptone- Azolectin-Tween and Tween 80 (TAT and T80) produced higher levels of Bacillus, estimated at 4.18 log CFU/g compared to growth on Modified letheen broth and Tween 80, which produced 3.97 log CFU/g (P < 0.05). Colony counts of B. cereus cells on MYP agar were significantly higher, than those on BACARA agar, showing an average of 4.25 log CFU/g versus 3.84 log CFU/g, respectively (P < 0.05). The growth of the strain mislabeled B. megaterium ATCC 6458 on B. cereus selective agars BACARA and MYP agar led us to further investigations. We identified bi-pyramidal crystals among colonies of the strain, and subsequent PCR identified the cry 1 gene, indicating that strain was actually B. thuringiensis subps. kurstaki.

Kinetics of continuous production of β‐carotene in an airlift bioreactor

AbstractThe focus in the present work was on the continuous β‐carotene production from free fatty acids supplied by the soap stock as a sole carbon source for Rhodotorula rubra cells in an airlift bioreactor. The effects of dilution rate on the cell growth and β‐carotene production were studied by both freely suspended‐ and immobilized‐cell systems in the chemostat operations. The results showed that the immobilization of the cell on natural sponge of loofah significantly increased the optimum and maximum dilution rates from 0.0375 to 0.05 hr−1 and 0.055 to 0.150 hr−1, respectively. The immobilization process has practical role in balancing of cell proliferation in the matrix and wash out the grown cells containing β‐carotene from the bioreactor where β‐carotene productivity was enhanced from 5.4 to 23.4 mg L−1 hr−1 using the immobilized cell technology.

A new method for fast extraction and determination of chlorophylls in natural water.

Algae collection and chlorophyll extraction are two troublesome steps in the traditional methods used for the determination of chlorophyll concentration in natural water. A new method was established in this study for fast collection and extraction of chlorophyll. Based on our results, the optimum centrifugation condition for collecting algae was determined as: 5000 g for 15 min at 4 °C, and the optimum dilution ratio of dimethyl sulfoxide: 90% acetone was 1:4. The specific steps were as follows: the algae in water samples were collected by centrifugation at 5000 g at 4 °C for 15 min. The precipitated algae were suspended with 2 mL DMSO. Then the sample was transferred to a 15 mL centrifuge tube, and the tube was incubated at 65 °C for 1-2 h in the dark until the sample turned white. After cooling, the chlorophyll extract was diluted with 8 mL 90% acetone, before centrifugation at 5000 g for 5 min. The absorbance values of the supernatants at 750, 664, 647 and 630 nm were used for the calculation of chlorophyll concentrations by the trichromatic equations. This new method saved the filter cost, simplified the extraction process, improved the algae acquisition efficiency, and accelerated the chlorophyll extraction rate.

Overall development of a bioprocess for the outdoor production of Nannochloropsis gaditana for aquaculture

In this paper, a systematic methodology is shown for the scaling-up of Nannochloropsis gaditana production for aquaculture uses. First, an adequate culture medium was developed, prepared using fertilizers instead of pure chemicals. Subsequently, the performance of N. gaditana was modelled as a function of average irradiance; this model being validated in continuous culture experiments. The model was used to determine the optimal dilution rate as well as the expected biomass concentration and productivity at optimal conditions. Finally, outdoor experiments were performed to confirm the model's validity and to determine optimal conditions at real production step. Biomass productivity values of up to 0.08 g L−1 day−1 were obtained at an optimal dilution rate of 0.25 per day in 0.2 m wide flat-panel reactors using culture medium containing 0.4 g/L NO−3 and 0.034 g/L·PO4−3. Fish trials with Atlantic Salmon demonstrated that the inclusion of produced biomass into fed increase the final weight up to 5%, thus confirming the adequacy of the biomass produced for aquaculture uses. The growth model and the scaling-up strategy proposed here are necessary to develop real industrial-scale processes capable of supplying microalgal biomass to the aquaculture markets, which in turn require a guaranteed supply and quality of the raw materials provided.

The Effects of Micromixing Two Solutions of Two Concentrations in a Two Tier PDMS Micromixer

Micromixing technology has drastically advanced in the past few decades. Micromixers are one of the elements in integrated microfluidic systems for chemical, analytical chemistry, pharmaceutical, and biological applications. In this study, two tier micromixer was used to mix and dilute two solutions of similar and different concentration in order to investigate performance of micromixer’s mixing. This paper presents the fabrication of a designed micromixer using polydimethylsiloxane (PDMS) and vinyl tape methods which reduce time, cost and complexity of prototyping. The serpentine structure of the microchannels was designed to enhance both mixing and dilution. Two types of food dyes and distilled water were used to investigate the mixing performance of the micromixer followed by spectrophotometry analysis. It is observed that the single dye solution and distilled water shows better mixing performance compared to the micromixing of two dye solutions which was supported by the diffusion theory. 2.00 ml/min was the optimum flow rate that allow optimum mixing and dilution between two different concentrated liquids.

Scenedesmus obliquus mediated brewery wastewater remediation and CO2 biofixation for green energy purposes

Microalgae can be used for wastewater bioremediation with simultaneous CO2 biofixation producing valuable biomass.Wastewater from a brewery was treated using the Scenedesmus obliquus microalga in bubble-column photobioreactors (PBRs). The PBRs were fed with ambient air and the effect of a 10% (v/v) brewery CO2 supplement was studied. The PBRs were inoculated and a range of mean hydraulic residence time (HRT) values were tested (2.1–10.4 days). The maximum ash-free dry weight (AFDW) biomass productivity was obtained for a HRT of 3.5 days (0.29 d−1 dilution rate; 0.2 g L−1 d−1 (in terms of AFDW). The highest pollutant removal efficiencies were 92.9 and 88.5% for ammonia and total nitrogen, respectively, 40.8% for phosphorus, and 61.9% for COD. Except a dilution rate of 0.48 d−1 trial, the treated wastewater always met the Portuguese legislation quality standard for discharge into natural water bodies. Aiming to simultaneously maximize biomass volumetric productivity, CO2 biofixation rate and wastewater treatment efficiency, while minimizing residence time, 0.29 d−1 represents the optimal dilution rate value.The potential of the produced Scenedesmus obliquus biomass was evaluated for the generation of biohydrogen through dark fermentation with Enterobacter aerogenes, and of bio-oil, bio-char and bio-gas through a pyrolysis process. The yields obtained were 67.1 mL H2 g−1 (in terms of volatile solids - VS) for bioH2 and 64, 30 and 6% for bio-oil, bio-char and bio-gas, respectively (dry mass content (%) calculated over freeze dryer biomass basis).

Phase equilibria, interfacial, and bulk compositions of microemulsions containing short-chain alcohols studied by an optimum microemulsion dilution method

An optimum microemulsion dilution method was presented to explore the phase behavior of imidazolium type surfactant 1-alkyl-3-methylimidazolium bromide (C12mimBr, C14mimBr, and C16mimBr)/short-chain alcohol (butan-1-ol, propan-1-ol)/oil/water systems. The dilution method was obtained based on the geometrical consideration in the composition tetrahedron phase diagram. Two dilution equations were deduced. The solubilities of the alcohol in oil and water phases were calculated, respectively. The composition of the surfactant-and-alcohol interfacial layer, represented by the mass fraction of alcohol in the interfacial layer, A S , and the maximum solubilization ability of the microemulsion phase, SP*, were also obtained.

Assessment of metal ion concentration in water with structured feature selection

We propose a cost-effective system for the determination of metal ion concentration in water, addressing a central issue in water resources management. The system combines novel luminometric label array technology with a machine learning algorithm that selects a minimal number of array reagents (modulators) and liquid sample dilutions, such that enable accurate quantification. The algorithm is able to identify the optimal modulators and sample dilutions leading to cost reductions since less manual labour and resources are needed. Inferring the ion detector involves a unique type of a structured feature selection problem, which we formalize in this paper. We propose a novel Cartesian greedy forward feature selection algorithm for solving the problem. The novel algorithm was evaluated in the concentration assessment of five metal ions and the performance was compared to two known feature selection approaches. The results demonstrate that the proposed system can assist in lowering the costs with minimal loss in accuracy.

Use of Dual-Force Aggregation as a Multiplexed, Rapid Point-of-Care Screening Method for White Blood Cell Counting from Whole Blood Samples.

Enumeration of blood cells is an integral metric for evaluating patient health and can be used to screen for a wide range of diseases and conditions. Conventional methods rely on large, expensive, and complicated instrumentation that requires trained technicians and is not amenable to point-of-care analysis. This work demonstrates the use of a multiplexed, bead-based assay for both rapid white blood cell (WBC) count screening and accurate, multiplexed WBC counts for point-of-care analysis. Blood samples were lysed and diluted before being incubated with silica-coated magnetic particles under chaotropic conditions, a rotating magnetic field, and a source of agitation. The resulting bead aggregation was imaged and correlated to a known WBC count. After establishing standard curves, the WBC count for 18 whole blood samples were determined by this method and compared to values obtained conventionally. When the optimal dilution factor for lysis of whole blood samples was established, 17 of 18 samples (94.4%) were correctly screened and categorized as having high, typical, or low WBC count, while 14 of 18 samples were within 16% of the reported clinical values. The developed system provides analysis of 13 samples in <3 min with a total analysis time of approximately 10 min (including incubation and dilution) and represents comparable throughput to conventional instrumentation, while providing point-of-care capability with reduced size (14 × 21 × 14 cm) and simplicity. This work demonstrates the potential for a multiplexed, bead-based assay to be used as a rapid, point-of-care screening method for WBC counting from whole blood samples.

Pretreatment of brewery effluent to cultivate Spirulina sp. for nutrients removal and biomass production.

Due to the low concentration of nitrate and high contents of organics, brewery effluent was not suitable for the cultivation of Spirulina sp. This work changed the nutrient profile of brewery effluent effectively by dilution, addition of nitrate, and anaerobic digestion. The result showed that the optimum dilution rate and NaNO3 addition for brewery effluent were 20% and 0.5 g/L, respectively. Spirulina sp. grown in pretreated brewery effluent produced 1.562 mg/L biomass and reduced concentrations of nutrients to reach the permissible dischargeable limits. In addition, Spirulina sp. grown in pretreated brewery effluent had much higher protein content and oil content. So the appropriate treatment converted brewery effluent into a nutrient balanced medium for algae cultivation and alleviated the potential environmental problems. Pretreatment procedure developed in this work is an effective way to realize the sustainable utilization of brewery effluent and produce algal biomass with valuable nutrients.

Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.

Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa.

Objective(s):Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.Materials and Methods:We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The transformants were expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.Results:The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.Conclusion:Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.

Platelet Detection in Unaltered Whole Blood

Abstract Platelets are non-nucleated cellular fragments circulating in the peripheral blood, derived from megakaryocytes. They are critical in the maintenance of hemostasis. Alterations in their function are associated with a range of clinical conditions. Using flow cytometry, light scatter signals from platelets typically overlap signals from red blood cells (RBC). Since RBC outnumber platelets by more than an order of magnitude in whole blood, platelets are typically studied by depleting samples of RBCs by centrifugation methods or selective RBC cell lysis. These sample preparation procedures require manipulations that may affect platelet health and function, and result in loss of cells. Additionally, lysed samples require labeling of the platelets with a fluorescent marker to adequately resolve the platelets from debris/noise. We present a novel technique for resolving leukocytes and platelets from RBC in whole blood using orthogonal light scatter from 2 lasers. This method exploits the differences in light-scattering properties between RBCs and platelets and white blood cells (WBC). Hemoglobin readily absorbs 405 nm laser light, reducing the scatter signal from RBC relative to WBCs and platelets, resulting in a reproducible scatter pattern when analyzing human whole blood with both blue (488 nm) and violet (405 nm) side scatter. Using this approach, a distinct platelet population can be discerned in unaltered whole blood, requiring only dilution of whole blood in buffer. Combining this approach with acoustic cytometry allows great flexibility in determining optimal dilution and sample collection rates for minimal coincidence and rapid analysis. For research use only. Not for use in diagnostic procedures.

IMPROVEMENT OF THE QUALITY CONTROL OF ELISA TESTING FOR THE LABORATORY CONFIRMATION OF MEASLES AND RUBELLA INFECTIONS AT THE STAGE OF THE MEASLES/ RUBELLA ELIMINATION PROGRAM

To estimate ELISA serological studies results of IgM and IgG specific Measles and Rubella Viruses (MRV) antibodies detection the “in-house” laboratory controls (ILC) including the specific markers of MRV infections were for the first time commercially prepared by the Vector Best PLC (Russia): “Measles-IgM, ser.1”, “Measles-IgM, ser.2”, “Rubella-IgM”, Measles-IgG” and “Rubella-IgG”. This task was realized under the special Executive Order of the Government of Russia N 523-r, 2014, April, 4. According to passport characteristics ILC samples are the lyophilized human sera, inactivated by heating (1 hour at 56°C) and stabilized by the mixture of sucrose (5%) and ProClin-3000 as the conservation agent. Samples are free of HBs Ag, anti-HVC, T.Pallidum, HIV-1/2, HIV-1Ag р24.The aim of the study was to evaluate the possibility of using the ILC for detection of the MRV IgM and IgG antibodies by ELISA with commercial ELISA kits used in Russia and CIS countries. In the process of detecting the specific activity of “Measles-IgM, ser.1”, “Measles-IgM, ser.2” and “Rubella-IgM” by ELISA kits of different formats (Vector Best, EcoLab and Siemens Companies) the statistically different results were received (p 0.05). The optical density (OD) values of IgM in the “Measles-IgM, ser.1” and “Measles-IgM, ser.2” ILC, obtained by ELISA “VectoMeasles IgM” (Vector Best) were significantly higher than those obtained by ELISA IgEnzygnost®Anti-MeaslesVirus/IgМ. These values consisted for the ser. 1–1.33±0.02 о.u. vs. 0.18±0.01 о.u. (р 0.05) and for the ser. 2–2.83±0.03 о.u. vs. 0.7±0.02 о.е. (р 0.05) in the Vector Best and Siemens ELISA kits correspondently. In the “Rubella-IgM” ILC the OD values of the specific IgM by the “ELISA-Rubella IgM” EcoLab were also higher than those obtained by IgEnzygnost®Anti-RubellaVirus/IgМ ELISA kit. These values consisted 2.92±0.04 о.u. vs. 0.88±0.03 о.u. (р 0.05) in EcoLab and Siemens ELISA test-systems correspondently. In the studies when the ILC working solution parameters for ELISA of “capture” and “indirect” formats were evaluated the statistic reliability and high information capability of the mathematic models of the OD value changes vs. the degree of dilution of the initial ILC were determined. For the “capture” and “indirect” formats the determination coefficient (R2) consisted 97.34 and 99.29 correspondently, the Fisher criteria (F) — 219.62 and 556.55 correspondently, the significance level (p) — 0.05 for both formats. The evaluated optimal working dilution degrees of the IgM-containing ILC make possible to exclude the influence of the IgG antibodies as the non-target marker in the ILC. On the other hand, taking into account the format of the used ELISA kit the optimal working dilution degrees give the possibility to obtain the desirable 2–3 “cut-off” values of the specific marker. Moreover, the issue of the target marker quantitative content in ILC is discussed in the study. In order to improve the quality of the commercial MRV IgM and IgG ELISA test-systems the joint studies with those who develop and produce these systems are desirable to be organized.

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Restriction capillaries as an innovative mixing unit for intermediate mobile phase exchange in multidimensional analysis

A novel mixing unit is proposed for the serial coupling of orthogonal columns to analyze polar and non-polar compounds in a single run. The principle relies on the isolation of unretained peaks eluting from a first dimension column in a sample loop, before directing them to a second column for separation. Since the mobile phases employed in highly orthogonal separations are not directly compatible, a mixing unit is required to alter the mobile phase composition before executing the second dimension separation. The mixing unit proposed in this work is based on the use of two restriction capillaries with different flow resistances to dilute the mobile phase eluting from the first dimension with a solvent appropriate for the second dimension separation. The restriction capillaries are implemented in an ultra-high performance liquid chromatography set-up using three high-pressure switching valves and two T-pieces. It is demonstrated that the dilution ratio can be adequately predicted using the law of Hagen-Poiseuille and can be adjusted easily by changing the dimensions of the restriction capillaries. The dilution volume required to obtain acceptable recoveries is investigated and the use of different column diameters in the first and second dimension is proposed to increase the sensitivity of the analysis. Under optimum dilution conditions, recoveries ranging between 82% and 99% are always obtained, while repeatability values are excellent. The proof-of-concept of the different set-ups is demonstrated for the separation of 20 pharmaceuticals with log D-values ranging between −5.75 and 4.22.

A PCR-free point-of-care capacitive immunoassay for influenza A virus

This article describes a highly sensitive and specific capacitive immunosensor for rapid, low cost and simple-to-use detection of virus particles from clinical swab samples. An inhomogeneous AC electric field is applied on sensor electrodes. This induces positive dielectrophoresis that attracts virus particles to the sensor electrodes. As a result, rapid and sensitive detection of influenza A virus is accomplished without the need for nucleotide isolation and amplification. The same AC signal is used to detect the binding of virus particle to the sensor surface immobilized with the antibody probe. The assay is highly suitable for point-of-care use. When testing clinical swab samples, the response of samples at various dilutions is analyzed, and an optimal dilution is found and used for subsequent blind tests of clinical swab samples. Analytical experiments on standard influenza virus sample demonstrate a limit of detection of 0.25 pg⋅mL−1. Other figures of merit include (a) an assay time of 30 seconds; (b) a diagnostic sensitivity of 90%; and (c) a specificity of 70%. Blind tests are conducted for a panel of twenty nasal swab samples, and the results are in good agreement with those by using the commercial reverse transcription polymerase chain reaction.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis.

The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

Improving bioethanol production by Scheffersomyces stipitis using retentostat extractive fermentation at high xylose concentration

The improvement of yields and productivity for second-generation ethanol (2G) production as well as a better understanding of industrial yeasts and processes are required. Nevertheless, a process to produce 2G ethanol efficiently from lignocellulosic C5-sugars has not yet been proposed. Scheffersomyces stipitis presents good fermentation performance, promising to be a potential biocatalyst for converting C5-sugars to ethanol. In order to evaluate yeast performance, experiments were carried out in a retentostatic extractive vacuum system (REV) with total cell retention, controlled substrate feeding (at different xylose concentrations 100–200gL−1) and simultaneous ethanol production and extraction. During REV at 100gL−1, it was possible to achieve an efficient ethanol production. The optimum dilution rate was 0.025h−1. To avoid stress effects to the yeast metabolism, the ethanol was continuously extracted. Low ethanol concentrations were kept inside the fermenter (25–35gL‐1), resulting in higher yields and productivity when compared with batch, fed-batch and continuous cultures.