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Culture Methods (culture + methods)
Selected AbstractsComparison of Three Culture Methods for the Intensive Culture of Northern Quahog Seed, Mercenaria mercenariaJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2001Timothy J. Pfeiffer A number of approaches have been utilized for growing bivalve hatchery seed (1 mm) to a size suitable for field planting (< 8 mm) but few have been directly compared. This study evaluated the growth and survival of northern quahog seed in three different culture systems and two different stocking densities. The three systems were: 1) a stacked-tray unit with downward water flow; 2) traditional upweller culture units with water flowing upward without seed bed expansion; and 3) upweller culture units with water flowing upward at fluidization velocities to provide seed bed expansion. The two stocking densities were 1.0 and 3.0 g whole wet weight clam/cm2 respectively. During each trial period the seed clams were fed a 1% daily ration (% dry weight algae per wet weight clam per day) of the cultured diatom Chaetoceros muelleri. After 14 d of culture at the 1.0 g whole wet weight/cm2 stocking density, seed clams (4.4 ± 0.6 mm initial shell length) under fluidized-flow condition exhibited better growth (0.54/d), and a greater final shell length (5.9 ± 1.0 mm). At the high density stocking conditions, after 28 d of culture, seed clams (4.2 ± 0.6 mm initial shell length) in the fluidized-flow culture conditions again exhibited better growth rate (0.031/d) and a greater final shell length (6.0 ± 1.0 mm). The preliminary evaluation of fluidized-flow for seed clam culture in land-based nurseries indicates its potential as a suitable alternative to raceway, downwelling, or traditional forced-flow culture methods. [source] Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a reviewFEMS MICROBIOLOGY REVIEWS, Issue 5 2005Uta Gasanov Abstract Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked. [source] Comparison of different methods of bacterial detection in blood componentsISBT SCIENCE SERIES: THE INTERNATIONAL JOURNAL OF INTRACELLULAR TRANSPORT, Issue 1 2009M. Schmidt Background, Over the last two decades, the residual risk of acquiring a transfusion-transmitted viral infection has been reduced to less than 1 : 1 000 000 via improvements in different techniques (e.g. donor selection, leuco-depletion, introduction of 3rd or 4th generation enzyme-linked immunosorbent assays and mini-pool nucleic acid testing (MP-NAT). In contrast, the risk for transfusion-associated bacterial infections has remained fairly stable, and is estimated to be in a range between 1 : 2000 and 1 : 3000. Platelets are at an especially higher risk for bacterial contamination, because they are stored at room temperature, which provides good culture conditions for a broad range of bacterial strains. To improve bacterial safety of blood products, different detection systems have been developed that can be divided into culture systems like BacT/ALERT or Pall eBDS, rapid detection systems like NAT systems, immunoassays and systems based on the FACS technique. Culture systems are used for routine bacterial screening of platelets in many countries, whereas rapid detection systems so far are mainly used in experimental spiking studies. Nevertheless, pathogen-reduction systems are currently available for platelet concentrates and plasma, and are under investigation for erythrocytes. Methods, In this review, the functional principles of the different assays are described and discussed with regard to their analytical sensitivity, analytical specificity, diagnostic sensitivity, diagnostic specificity and clinical efficiency. The detection methods were clustered into three groups: (i) detection systems currently used for routine screening of blood products, (ii) experimental detection systems ready to use for routine screening of blood products, and (iii) new experimental detection systems that need to be investigated in additional spiking studies and clinical trials. Results, A recent International Society of Blood Transfusion international forum reported on bacterial detection methods in 12 countries. Eight countries have implemented BacT/ALERT into blood donor screening, whereas in three countries only quality controls were done by culture methods. In one country, shelf-life was reduced to 3 days, so no bacterial screening was implemented. Screening data with culture methods can be used to investigate the prevalence of bacterial contamination in platelets. Differing results between the countries could be explained by different test definitions and different test strategies. Nevertheless, false-negative results causing severe transfusion-related septic reactions have been reported all over the world due to a residual risk of sample errors. Rapid screening systems NAT and FACS assays have improved over the last few years and are now ready to be implemented in routine screening. Non-specific amplification in NAT can be prevented by pre-treatment with Sau3AI, filtration of NAT reagents, or reduction of the number of polymerase chain reaction cycles. FACS systems offer easy fully automated handling and a handling time of only 5 min, which could be an option for re-testing day-5 platelets. New screening approaches like immunoassays, detection of bacterial adenosine triphosphate, or detection of esterase activity need to be investigated in additional studies. Conclusion, Bacterial screening of blood products, especially platelets, can be done with a broad range of technologies. The ideal system should be able to detect one colony-forming unit per blood bag without a delay in the release process. Currently, we are far away from such an ideal screening system. Nevertheless, pathogen-inactivation systems are available, but a system for all blood components will not be expected in the next few years. Therefore, existing culture systems should be complemented by rapid systems like NAT or FACS especially for day-5 platelets. [source] A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNAJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007A. Lorusso Abstract Aims:, The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. Methods and Results:, A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 101 DNA copies per 10 ,l of plasmid template and 6·5 × 100 colour changing units of reference strain Ba/2. Conclusions:, The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2,3 h compared with at least 1 week). Significance and Impact of the Study:, The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies. [source] Improvements in the production of bacterial synthesized biocellulose nanofibres using different culture methodsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2010Amir Sani Abstract This review summarizes previous work that was done to improve the production of bacterial cellulose nanofibres. Production of biocellulose nanofibres is a subject of interest owing to the wide range of unique properties that makes this product an attractive material for many applications. Bacterial cellulose is a natural nanomaterial that has a native dimension of less than 50 nm in diameter. It is produced in the form of nanofibres, yielding a very pure cellulose product with unique physical properties that distinguish it from plant-derived cellulose. Its high surface-to-volume ratio combined with its unique properties such as poly-functionality, hydrophilicity and biocompatibility makes it a potential material for applications in the biomedical field. The purpose of this review is to summarize the methods that might help in delivering microbial cellulose to the market at a competitive cost. Different feedstocks in addition to different bioreactor systems that have been previously used are reviewed. The main challenge that exists is the low yield of the cellulosic nanofibres, which can be produced in static and agitated cultures. The static culture method has been used for many years. However, the production cost of this nanomaterial in bioreactor systems is less expensive than the static culture method. Biosynthesis in bioreactors will also be less labour intensive when scaled up. This would improve developing intermediate fermentation scale-up so that the conversion to an efficient large-scale fermentation technology will be an easy task. Copyright © 2009 Society of Chemical Industry [source] A prevalence survey for zoonotic enteric bacteria in a research monkey colony with specific emphasis on the occurrence of enteric YersiniaJOURNAL OF MEDICAL PRIMATOLOGY, Issue 1 2001Stephen J. Vore Transmissible pathogenic and opportunistic zoonotic enteric bacteria comprise a recognized occupational health threat to exposed humans from non-human primates (NHPs). In an effort to evaluate the occurrence of selected enteric organisms with zoonotic and biohazard potential in a research colony setting, we performed a prevalence study examining 61 juvenile and young adult rhesus macaques participating in a transplant immunology project. Primary emphasis was directed specifically to detection of pathogenic enteric Yersinia, less well-documented and reported NHP pathogens possessing recognized significant human disease potential. NHPs were surveyed by rectal culture during routine health monitoring on three separate occasions, and samples incubated using appropriate media and specific selective culture methods. Enteric organisms potentially transmissible to humans were subcultured and identified to genus and species. Significant human pathogens of the Salmonella/Shigella, Campylobacter, and enteric Yersinia groups were not isolated throughout the survey, suggesting prevalence of these organisms may generally be quite low. [source] Comparison of Three Culture Methods for the Intensive Culture of Northern Quahog Seed, Mercenaria mercenariaJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2001Timothy J. Pfeiffer A number of approaches have been utilized for growing bivalve hatchery seed (1 mm) to a size suitable for field planting (< 8 mm) but few have been directly compared. This study evaluated the growth and survival of northern quahog seed in three different culture systems and two different stocking densities. The three systems were: 1) a stacked-tray unit with downward water flow; 2) traditional upweller culture units with water flowing upward without seed bed expansion; and 3) upweller culture units with water flowing upward at fluidization velocities to provide seed bed expansion. The two stocking densities were 1.0 and 3.0 g whole wet weight clam/cm2 respectively. During each trial period the seed clams were fed a 1% daily ration (% dry weight algae per wet weight clam per day) of the cultured diatom Chaetoceros muelleri. After 14 d of culture at the 1.0 g whole wet weight/cm2 stocking density, seed clams (4.4 ± 0.6 mm initial shell length) under fluidized-flow condition exhibited better growth (0.54/d), and a greater final shell length (5.9 ± 1.0 mm). At the high density stocking conditions, after 28 d of culture, seed clams (4.2 ± 0.6 mm initial shell length) in the fluidized-flow culture conditions again exhibited better growth rate (0.031/d) and a greater final shell length (6.0 ± 1.0 mm). The preliminary evaluation of fluidized-flow for seed clam culture in land-based nurseries indicates its potential as a suitable alternative to raceway, downwelling, or traditional forced-flow culture methods. [source] Evaluation of quantitative PCR and culture methods for detection of house dust fungi and streptomycetes in relation to moisture damage of the houseLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2008U. Lignell Abstract Aims:, Microbial concentrations in vacuumed house dust samples (n = 71) were analysed by culture and quantitative polymerase chain reaction (qPCR) methods and their association with extent of moisture damage in the house was studied. Methods and Results:, Microbial concentrations measured by qPCR correlated with concentrations obtained by culture method, but were orders of magnitude higher. qPCR also had better sensitivity. Concentrations of several microbes in house dust, determined with qPCR, were associated with the extent of moisture damage in the house. This association was strongest for Penicillium brevicompactum, one of the fungi detected in highest concentrations by qPCR. Furthermore, house dust concentrations of Wallemia sebi, Trichoderma viride, Cladosporium sphaerospermum, Eurotium amstelodami and the combined assay group for Penicillium spp., Aspergillus spp. and Paecilomyces variotii were significantly associated with the extent of the moisture damage. Conclusion:, These species or assay groups could probably be used as indicators of moisture damage in the house. Significance and Impact of the Study:, This finding indicates the benefits of the qPCR method, which is sensitive enough to reveal the differences in microbial concentrations of house dust between moisture-damaged and undamaged houses. [source] Improved bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken meat samplesLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2005S. Fukuda Abstract Aims:, To evaluate an improved bioluminescent enzyme immunoassay (BEIA) using biotinylated firefly luciferase for the rapid detection of Salmonella in naturally contaminated chicken meat samples. Methods and Results:, Capture agents and lipopolysaccharide (LPS) extraction reagents for Salmonella were investigated to improve the sensitivity of the BEIA. Also, the use of Oxoid SPRINT (Simple Pre-enrichment and Rapid Isolation New Technology) as a pre-enrichment and selective medium for 26-h BEIA detection of Salmonella in chicken meat samples was examined. The use of polymyxin B as a capture agent on solid support and 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) for extraction of the LPS facilitated sensitive detection of Salmonella. Of 120 chicken meat samples, 25 samples were positive using the improved BEIA with the SPRINT and 25 samples were positive using the SPRINT followed by the standard isolation methods. Conclusions:, The improved BEIA, in which polymxin B was used as a capture agent and CHAPS was used for extraction of the antigen, had a sensitivity of 96·0% and a specificity of 98·9% for the detection of Salmonella in chicken meat. Significance and Impact of the Study:, The improved BEIA combined with the SPRINT medium for the detection of Salmonella in chicken meat samples produced comparable results to the culture methods in 26 h. [source] Rapid detection of Haemophilus influenzae by hel gene polymerase chain reactionLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2003M.C. Yadav Abstract Aims: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. Methods and Results: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65·9%) of 91 samples in contrast to 62 (68·12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. Conclusions: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. Significance and Impact of the study: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods. [source] Vector transmission of Bartonella species with emphasis on the potential for tick transmissionMEDICAL AND VETERINARY ENTOMOLOGY, Issue 1 2008S. A. BILLETER AbstractBartonella species are gram-negative bacteria that infect erythrocytes, endothelial cells and macrophages, often leading to persistent blood-borne infections. Because of the ability of various Bartonella species to reside within erythrocytes of a diverse number of animal hosts, there is substantial opportunity for the potential uptake of these blood-borne bacteria by a variety of arthropod vectors that feed on animals and people. Five Bartonella species are transmitted by lice, fleas or sandflies. However, Bartonella DNA has been detected or Bartonella spp. have been cultured from numerous other arthropods. This review discusses Bartonella transmission by sandflies, lice and fleas, the potential for transmission by other vectors, and data supporting transmission by ticks. Polymerase chain reaction (PCR) or culture methods have been used to detect Bartonella in ticks, either questing or host-attached, throughout the world. Case studies and serological or molecular surveys involving humans, cats and canines provide indirect evidence supporting transmission of Bartonella species by ticks. Of potential clinical relevance, many studies have proposed co-transmission of Bartonella with other known tick-borne pathogens. Currently, critically important experimental transmission studies have not been performed for Bartonella transmission by many potential arthropod vectors, including ticks. [source] Tissue culture methods to study neurological disorders: Establishment of immortalized Schwann cells from murine disease modelsNEUROPATHOLOGY, Issue 1 2003Kazuhiko Watabe Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann,Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npcnih/npcnih, npcnih/+), myelin P0 protein-deficient mice (P0,/,, P0+/,) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1Fcr/+) for 8,10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders. [source] Microchip, reverse transcription-polymerase chain reaction and culture methods to detect enterovirus infection in pediatric patientsPEDIATRICS INTERNATIONAL, Issue 1 2006LON-YEN TSAO Abstract Background: Enterovirus infection usually presents with mild and self-limited illness in children. However, Enterovirus type 71 can be characterized by neurotropism and may cause severe illness or even sudden death. Early detection of the virus will allow a physician to provide intensive or aggressive intervention. The purpose of the present study was to compare sensitivity of two innovative laboratory methods, that is, the DR.EV microchip method (DR. Chip Biotechnology, Shin-Tsu, Taiwan) and the reverse transcription-polymerase chain reaction (RT-PCR) method following conventional virus culture in detecting enterovirus infection in pediatric patients with herpangina or hand,foot,mouth disease. Methods: A total of 87 children (age range: 1,8 years) were enrolled because of typical clinical findings of herpangina and hand,foot,mouth disease. Two hundred children selected after a careful clinical history review and physical examinations, were included as controls. All of these children had at least throat swab and rectal swab specimens taken and tested for evidence of enterovirus infection by microchip, RT-PCR and virus culture methods. In addition, 21 patients also had cerebrospinal fluid (CSF) specimens taken to test for possible central nervous system involvement. Result: The test results obtained from the 200 healthy kindergarten children were all negative for enteroviral infection by these three methods. Among the 87 test patients, the positive rates for throat swab, rectal swab and CSF by DR.EV chip, RT-PCR and virus culture were 71%, 68%, and 45% (throat swab); 66%, 61%, and 33% (rectal swab); and 52%, 29%, and 5% (CSF), respectively. There was no significant difference in the positive rates between the DR.EV chip and the RT-PCR methods (P > 0.1) on all types of specimens. However, statistically significant differences in positive rates were noted between the DR.EV chip and the conventional virus culture methods on all types of specimens (P < 0.001). Sensitivity of the microchip, RT-PCR and virus culture methods, was 82%, 72%, and 53%, respectively. Conclusion: The DR.EV chip method yielded a statistically higher positive rate and faster test results than the conventional viral culture method. [source] Molecular and agronomic evaluation of wheat doubled haploid lines obtained through maize pollination and anther culture methodsPLANT BREEDING, Issue 4 2003J. Guzy-Wrobelska Abstract Although maize pollination (MP) and anther culture (AC) are alternative techniques widely used for wheat doubled haploid (DH) production, there is only limited information on the attributes of the plant materials produced through both methods. This study was conducted to evaluate genetic fidelity, transmission of parental gametes, and to compare field performance of DH populations produced by the MP and AC methods from the F1s of two crosses between spring bread wheat cultivars. The DH populations were compared to single seed descent (SSD) lines created from the same crosses. In total, 76 MP and 122 AC lines of the cross between cultivars of divergent origin were subjected to RAPD and AFLP analysis. Only changes in AFLP banding patterns, at similarly low frequencies, 0.18% (MP) and 0.21% (AC), were detected. The frequency of the DH lines affected by the variation, 14.5% (MP) and 14.8% (AC), was similar in both populations. For most of the DH lines, variation in 1-2 loci only, out of several hundreds scored, was observed. A total of 14.3% (MP) and 22.2% (AC) marker loci showed the significant segregation distortion from the expected 1 : 1 ratio, but in at least one polymorphic locus the within-cultivar variation was responsible for the skewed segregation. The field performance of the corresponding MP and AC lines derived from two crosses confirmed the equivalency of both DH populations. In most of the traits analyzed, the MP and AC lines performed the same as the SSD populations created from the same crosses. No, or very small differences in means and ranges, were observed when the best 10% of the lines from all three methods were compared. Moreover, the best 10 % of the lines of the cross between Polish wheat cultivars adapted to the local environment performed significantly better for some traits than different groups of checks used in the study. [source] QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of ricePLANT BREEDING, Issue 1 2002Y. S. Kwon Abstract Anther culturability of rice is a quantitative trait controlled by nuclear-encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ,Milyang 23' and ,Gihobyeo'. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F2 populations, ,MG RI036'/,Milyang 23', and ,MG RI036';/,IR 36' were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F2 populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes. [source] Economic analysis of monosex culture of giant freshwater prawn (Macrobrachium rosenbergii De Man): a case studyAQUACULTURE RESEARCH, Issue 9 2006C Mohanakumaran Nair Abstract All-male monosex culture of Macrobrachium rosenbergii (De Man) has emerged as a popular practice in India, especially in the state of Andhra Pradesh. A study was conducted to compare the economics of all-male, mixed and all-female culture in 15 adjacent, rectangular ponds of 4000 m2 each by stocking juveniles previously reared in a nursery for 60 days. The experiment was conducted using a completely randomized design with three treatments; T1 (all male), T2 (mixed) and T3 (all female), and five replicates for a period of 5 months after the nursery phase. Statistical analysis showed highly significant (P<0.01) differences among the three types of culture. The cost of production was estimated and the economic feasibility of the culture methods was evaluated by cost-return and partial budgeting analysis. The average weight, productivity and specific growth rate were the highest for all male culture, being 80.92±2.41 g, 1532 kg ha,1 and 1.97±0.02 respectively. All-female culture registered significantly higher survival (89.16±0.77%) and the best apparent feed conversion ratio of 1.26±0.02. The economic analysis revealed that all-male monosex culture of M. rosenbergii was 63.13% and 60.20% more profitable than mixed and all-female cultures respectively. [source] Infection and fetal loss in the mid-second trimester of pregnancyAUSTRALIAN AND NEW ZEALAND JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Issue 3 2010Ben ALLANSON Introduction:, Chorioamnionitis is a common cause of second trimester pregnancy loss, usually due to ascending infection. This study investigates the prevalence and bacteriology of chorioamnionitis in cases of spontaneous pregnancy loss in previable gestations (16,22 weeks). Methods:, Fetal losses between 16- and 22-week gestation were identified from the institutional database over a three-year period. Cases with an autopsy were identified, pathology reports reviewed, and maternal features noted (clinical symptoms, blood count and vaginal culture results). Second trimester medical termination for fetal abnormality during the same time period served as controls for the confounding influence of labour. Results:, A total of 101 cases of spontaneous non-anomalous non-macerated fetal losses and 103 control cases of induced loss for fetal anomaly were identified. Median gestation of cases was 19 weeks (interquartile range (IQR) 17, 21) and of controls was 20 weeks (IQR 19, 21). Maternal white cell count was higher in cases (median 13.6 IQR 10.8, 16.6) than in controls (9.9 IQR 7.6, 11.5) (P < 0.01). Seventy-eight (77.2%) of 101 cases and no controls had histological chorioamnionitis. A fetal reaction was identified in 48.7% of cases with chorioamnionitis, and the frequency of fetal reaction increased as gestation advanced (5.3% at 16-week gestation vs 33.3% at 22-week gestation). In cases with chorioamnionitis 36/76 (47.4%) were culture positive, whereas 4/25 (16%) without chorioamnionitis were culture positive. Conclusion:, In otherwise normal fetuses, chorioamnionitis is a common finding in mid-trimester pregnancy loss. Routine culture methods have a low sensitivity for isolation of the causative micro-organisms. This inflammatory process seems to predate the onset of labour and appears a primary mechanism in the aetiology of such losses. [source] Dynamic culture of droplet-confined cell arraysBIOTECHNOLOGY PROGRESS, Issue 1 2010Elisa Cimetta Abstract Responding to the need of creating an accurate and controlled microenvironment surrounding the cell while meeting the requirements for biological processes or pharmacological screening tests, we aimed at designing and developing a microscaled culture system suitable for analyzing the synergic effects of extracellular matrix proteins and soluble environments on cell phenotype in a high-throughput fashion. We produced cell arrays deposing micrometer-scale protein islands on hydrogels using a robotic DNA microarrayer, constrained the culture media in a droplet-like volume and developed a suitable perfusion system. The droplet-confined cell arrays were used either with conventional culture methods (batch operating system) or with automated stable and constant perfusion (steady-state operating system). Mathematical modeling assisted the experimental design and assessed efficient mass transport and proper fluidodynamic regimes. Cells cultured on arrayed islands (500 ,m diameter) maintained the correct phenotype both after static and perfused conditions, confirmed by immunostaining and gene expression analyses through total RNA extraction. The mathematical model, validated using a particle tracking experiment, predicted the constant value of velocities over the cell arrays (less than 10% variation) ensuring the same mass transport regime. BrdU analysis on an average of 96 cell spots for each experimental condition showed uniform expression inside each cell island and low variability in the data (average of 13%). Perfused arrays showed longer doubling times when compared with static cultures. In addition, perfused cultures showed a reduced variability in the collected data, allowing to detect statistically significant differences in cell behavior depending on the spotted ECM protein. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Real-time polymerase chain-reaction detection of pathogens is feasible to supplement the diagnostic sequence for urinary tract infectionsBJU INTERNATIONAL, Issue 1 2010Lutz E. Lehmann OBJECTIVE To evaluate, in a prospective pilot study, the feasibility of identifying pathogens in urine using real-time polymerase chain reaction (PCR), and to compare the results with the conventional urine culture-based procedures. PATIENTS AND METHODS Severe urinary tract infections (UTIs) are frequent in critically ill patients in the intensive-care unit (ICU) and in outpatients, and thus the reliable and fast identification of the bacteria is mandatory, but routine urine culture is time-consuming and the therapeutic regimen is often calculated and not culture-based. The study included 301 prospectively collected urine samples from 189 patients with suspected UTI, based in a university hospital in 2005, and included outpatients and those in the ICU. Urine culture with Cled-, MacConkey- and malt extract agar of all samples was followed by microbiological identification of the pathogens in 98 samples with visible growth. In parallel, all samples were assessed using qualitative real-time PCR-based DNA detection and identification by labelled hybridization probes. RESULTS In all, 15 dipstick culture-negative samples showed positive pathogen DNA identification by PCR. By contrast, 17 PCR-negative samples showed detectable pathogens by culture, of which 10 were not detectable on PCR because the identified pathogens were not represented in the probe panel. The sensitivity and specificity for detecting contaminated samples was 0.90 and 0.87, respectively. Overall, 95% of the mono-infection pathogens and 57% of the multiple-infection pathogens were detected concordantly with both methods. CONCLUSION In this prospective pilot study PCR-based identification of pathogens was feasible for supplementing conventional culture methods for the diagnosis of UTI. The main advantage is the time saved in identifying the pathogens. The limited pathogen detection in multiple-infection-samples by PCR might be explained by competitive PCR amplification conditions. [source] Volatiles Released by a Streptomyces Species Isolated from the North SeaCHEMISTRY & BIODIVERSITY, Issue 7 2005Jeroen The North Sea Streptomyces strain GWS-BW-H5 was investigated by analyzing headspace extracts of agar-plate cultures (HE) or liquid cultures (LCE), obtained with a closed-loop stripping apparatus (CLSA), by GC/MS (Table,1). The volatile profile of the HE is dominated by the known volatiles (,)-geosmin (4) and 2-methyisoborneol (1). Small amounts of sesquiterpenes occur, which are present in a more-diverse structural variety and in higher quantities in the LCE. The different structures can be rationalized by few cationic intermediates along their biosynthetic pathway. The most-prominent difference between the two culture methods were the presence of the Me-branched , - and , -lactones 31,38, not previously reported from nature, in the LCE. Major components were 10-methyldodecan-5-olide (34), 10-methyldodec-2-en-4-olide (36), and 10-methyldodec-3-en-4-olide (38). The structures of all new lactones were verified by synthesis. Furthermore, more volatiles in higher amounts were produced by the liquid culture than by to the agar plate culture. Since 36 showed inhibitory growth effects against strain GWS-BW-H5, growth inhibition against twelve other strains isolated from the same habitat was tested. Antagonistic activity against four of the strains was observed, with a slightly higher threshold level than found for penicillin G, which was used in control experiments (Table,2). [source] | |||||||||||||||||||