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Culture Techniques (culture + techniques)
Kinds of Culture Techniques Selected AbstractsApplication for regenerative medicine of epithelial cell culture-vistas of cultured epitheliumCONGENITAL ANOMALIES, Issue 3 2006Hajime Inoue ABSTRACT This review describes culture techniques for the epithelial system as well as trends in the clinical application of cultured keratinocytes in our department and the possibility of applying the techniques to other organs. Cultured epithelium and cultured dermis in particular have considerably preceded regeneration of other organs in the field of regenerative medicine. Since 1988 we have grafted cultured keratinocytes by the Rheinwald-Green modified method in at least 500 patients with large skin defects. As a result of the establishment of a culture technique for individual patients, it is now possible to prepare enough regenerated epithelium to cover the body surface area of as many as 10 adult patients in approximately three weeks after collecting 1 cm2 of skin, and then remaining cultured keratinocytes can be cryo-preserved for two-stage dermatoplasty at another site. This procedure makes it possible to avoid frequent skin collection from the same patient and thereby improves patients' quality of life and activities of daily living. On the other hand, to solve the problem of regenerated epithelium shrinking and problems with graft efficiency on dermis defect lesion, we have developed a proteinase-resistant regenerated dermis by mixing a certain protein with a fibrin scaffold. Recently we also took the initiative in grafting hybrid-type regenerated trachea in an animal experiment by using the epithelial and dermal cell culture technique, and some results of the graft were obtained. [source] Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.FEMS MICROBIOLOGY ECOLOGY, Issue 1 2009Alisha Akya Abstract Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata. The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions. [source] Risk and control of waterborne cryptosporidiosisFEMS MICROBIOLOGY REVIEWS, Issue 2 2002Joan B. Rose Abstract Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen. [source] Direct DNA delivery into zebrafish embryos employing tissue culture techniquesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2001Raquel Sussman Abstract Summary: The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24,48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources. genesis 31:1,5, 2001. © 2001 Wiley-Liss, Inc. [source] Is Helicobacter pylori a True Microaerophile?HELICOBACTER, Issue 4 2006Stephanie Bury-Moné Abstract Background:, There is no general consensus about the specific oxygen and carbon dioxide requirements of the human pathogen Helicobacter pylori. This bacterium is considered a microaerophile and consequently, it is grown under atmospheres at oxygen tensions 5,19% and carbon dioxide tensions 5,10%, both for clinical and basic and applied research purposes. The current study compared the growth of H. pylori in vitro, under various gas atmospheres, and determined some specific changes in the physiology of bacteria grown under different oxygen partial pressures. Methods:, Measurements of bacterial growth under various conditions were carried out employing classical solid and liquid culture techniques. Enzymatic activities were measured using spectrophotometric assays. Results:,H. pylori and all the other Helicobacter spp. tested had an absolute requirement for elevated carbon dioxide concentrations in the growth atmosphere. In contrast with other Helicobacter spp., H. pylori can tolerate elevated oxygen tensions when grown at high bacterial concentrations. Under 5% CO2, the bacterium showed similar growth in liquid cultures under oxygen tensions from microaerobic (< 5%) to fully aerobic (21%) at cell densities higher than 5 × 105 cfu/ml for media supplemented with horse serum and 5 × 107 cfu/ml for media supplemented with ,-cyclodextrin. Evidence that changes occurred in the physiology of H. pylori was obtained by comparing the activities of ferredoxin:NADH (nicotinamide adenine dinucleotide) oxidoreductases of bacteria grown under microaerobic and aerobic atmospheres. Conclusions:,H. pylori is a capnophile able to grow equally well in vitro under microaerobic or aerobic conditions at high bacterial concentrations, and behaved like oxygen-sensitive microaerophiles at low cell densities. Some characteristics of H. pylori cells grown in vitro under microaerobic conditions appeared to mimic better the physiology of organisms grown in their natural niche in the human stomach. [source] Antibacterial efficacy of calcium hydroxide intracanal dressing: a systematic review and meta-analysisINTERNATIONAL ENDODONTIC JOURNAL, Issue 1 2007C. Sathorn Abstract Aim, To determine to what extent does calcium hydroxide intracanal medication eliminate bacteria from human root canals, compared with the same canals before medication, as measured by the number of positive cultures, in patients undergoing root canal treatment for apical periodontitis (teeth with an infected root canal system). Methodology, CENTRAL, MEDLINE and EMBASE databases were searched. Reference lists from identified articles were scanned. A forward search was undertaken on the authors of the identified articles. Papers that had cited these articles were also identified through the Science Citation Index to identify potentially relevant subsequent primary research. Review methods, The included studies were pre-/post-test clinical trials comparing the number of positive bacterial cultures from treated canals. Data in those studies were independently extracted. Risk differences of included studies were combined using the generic inverse variance and random effect method. Results, Eight studies were identified and included in the review, covering 257 cases. Sample size varied from 18 to 60 cases; six studies demonstrated a statistically significant difference between pre- and post-medicated canals, whilst two did not. There was considerable heterogeneity among studies. Pooled risk difference was ,21%; 95% CI: ,47% to 6%. The difference between pre- and post-medication was not statistically significant (P = 0.12). Conclusions, Calcium hydroxide has limited effectiveness in eliminating bacteria from human root canal when assessed by culture techniques. [source] The persistence of bifidobacteria populations in a river measured by molecular and culture techniquesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2009X. Bonjoch Abstract Aims:, To determine relative to faecal coliforms (FC) and sulfite-reducing clostridia (SRC), the environmental persistence of natural populations of Bifidobacterium spp. enumerated by culturing and quantitative polymerase chain reaction (q-PCR). Methods and Results:, Dialysis tubing containing river supplemented with overnight cultures of Bifidobacterium adolescentis (BA) and Bifidobacterium dentium (BD) or urban wastewater were suspended in a river for up to 10 days. At intervals, the contents of each dialysis tube were assayed using q-PCR assays for BA and BD, and selective culture media for FC, SRC, total bifidobacteria (TB), sorbitol-fermenting bifidobacteria (SFB) and cultivable BA. Mean summer T90 values were 251 h for SRC, 92 h for FC, 48 h for BA and BD by q-PCR, and 9 h for TB. Conclusions:,Bifidobacterium spp. was the population with the lowest persistence, showing seasonal differences in T90 when measured by culture techniques or by q-PCR. This difference in relative persistence is because of a longer persistence of molecular targets than cultivable cells. Significance and Impact of the Study:, The persistence of a viable bifidobacteria cells is shorter, but the longest persistence of molecular targets. This factor could be used for origin the faecal pollution in water for the development of microbial source tracking (MST). [source] Women with a recent history of early-onset pre-eclampsia have a worse periodontal conditionJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 3 2007Alina Kunnen Abstract Objective: Pre-eclampsia is a complication of pregnancy characterized by systemic vascular dysfunction and pathological changes in placental arteries. Growing evidence of chronic infection as an aetiological factor in vascular diseases prompted us to study maternal periodontal disease in subjects with early-onset pre-eclampsia (<34 weeks). Methods: A case,control study was carried out on 17 early-onset pre-eclamptic women and 35 controls with uncomplicated pregnancies in a period of 3,28 months postpartum. All were Caucasians. Full-mouth periodontal examinations were performed to determine the periodontal condition. Subgingival-plaque samples were analysed by anaerobic culture techniques for the presence of seven bacterial periodontal pathogens. Potential confounders as age, smoking, educational level and body mass index were determined. Results: Severe periodontal disease was found in 82% of the pre-eclamptic and in 37% of the control group (p=0.009). After adjusting for age, smoking and educational level, the odds ratio was 7.9 (95% CI: 1.9,32.8). The periodontopathic microorganism Micromonas micros was more prevalent in the case group (p=0.040) while Campylobacter rectus was more prevalent in the control group (p=0.047). Conclusion: These results indicate that Caucasian women with a recent history of early-onset pre-eclampsia have a worse periodontal condition, as compared with women with uncomplicated deliveries. [source] Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a reviewJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 12 2004Mariano Sanz Abstract Background: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified. Aim: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf. Summary: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing. Conclusion: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology. [source] Absence of a specific subgingival microflora in adults with Down's syndromeJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 11 2001W. Reuland-Bosma Abstract Background: Periodontal disease in Down's syndrome (DS) is generally characterized by a high degree of bone loss. Bone loss of 5 mm or more is observed in 70% of these subjects. Among DS subjects, considerable differences in disease progression occur. So far, no studies have been conducted in which specific properties of the subgingival microflora have been related to the condition observed. Aims: To investigate (1) the subgingival microflora in DS subjects and other mentally retarded (control) individuals which were matched to the utmost and (2) to investigate the subgingival microflora of a "low-risk" and a " high-risk" group formed in DS subjects. Material and Methods: 17 DS subjects and 17 control subjects were matched with respect to age, plaque level and bleeding on probing. In addition, the DS group was divided in a "low-risk" group (0,2 teeth lost due to periodontal disease n=6) and a "high-risk"group (6,13 teeth lost due to periodontal disease n=11). Prevalence and proportions of the putative periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Peptostreptococcus micros, Fusobacterium nucleatum and Campylobacter rectus in the subgingival plaque were determined using anaerobic culture techniques. No differences in the prevalence of distinct suspected periodontopathic bacteria and bacterial subgingival composition between the DS group and the control group could be established. Also no differences in the prevalence of the seven investigated microbial species between the "low-risk" and the "high-risk" group were observed. Conclusions: Because of the lack of differences in microflora between the DS group and the control group, a specific effect of the microbiological composition in the periodontal status of subjects with DS can be excluded in this population. Host factors constitute the more likely explanation of the differences observed in DS. Zusammenfassung Basis: Die parodontale Erkrankung beim Down Syndrom (DS) ist allgemein durch einen hohen Grad von Knochenverlust charakterisiert. Knochenverlust von 5 mm und mehr wird bei 70% der Personen beobachtet. Unter den DS Personen bestehen beträchtliche Differenzen in der Erkrankungsprogression. Bis heute sind keine Studien durchgeführt worden, in welchen die spezifischen Eingenschaften der subgingivalen Mikroflora in Beziehung zu den Bedingungen beobachtet wurden. Ziele: Untersuchung (1) der subgingivalen Mikroflora bei DS Personen und anderen mental retardierten (Kontrollen) Personen, die zu einer größten gemischt wurden und (2) der subgingivalen Mikroflora von Gruppen mit "geringem Risiko" und Gruppen mit "hohem Risiko" bei DS Personen. Material und Methoden: 17 DS Personen mit 17 Kontrollpersonen wurden im Hinblick auf Alter, Plaquemenge und Blutung auf Provokation eingeteilt. Zusätzlich wurde die DS Gruppe in "geringes Risiko" (0,2 Zähne infolge von parodontaler Erkrankung verloren, n=6) und in "hohes Risiko" (6,13 Zähne infolge parodontaler Erkrankung verloren, n=11) eingeteilt. Das Vorkommen und die Relationen von putativen parodontalen Pathogenen Actinobacillus actinomycetemcomitants, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Peptostreptococcus micros, Fusobacterium nucleatum und Campylobacter rectus in der subgingivalen Plaque wurden mit anaerober Kulturtechnik bestimmt. Ergebnisse: Es konnten keine Differenzen in der Prävalenz der bezeichneten parodontopathogenen Bakterien und der bakteriellen subgingivalen Zusammensetzung zwischen DS Gruppe und der Kontrollgruppe beobachtet werden. Auch zwischen der Gruppe "geringes Risiko" und "hohes Risiko" wurden keine Differenzen in der Prävalenz der 7 untersuchten Spezies beobachtet. Schlußfolgerungen: Weil keine Differenzen in der Mikroflora zwischen DS Gruppe und der Kontrollgruppe vorhanden sind, kann ein spezifischer Effekt der mikrobiologischen Zusammensetzung beim parodontalen Status der Personen mit DS in dieser Population ausgeschlossen werden. Für die Erklärung der Differenzen, die bei den DS Personen beobachtet werden, sind die Wirtsfaktoren mehr wahrscheinlich. Résumé Origine: La maladie parodontale lors du syndrôme de Down (DS) est généralement caractérisée par une importante perte osseuse. Cette perte osseuse atteint 5 mm ou plus chez 70% de ces malades. Parmi les sujets DS, des différences considérables dans la progression de cette maladie se manifestent. Aucune étude n'a encore été entreprise dans laquelle la microflore sous-gingivale a été mise en relation avec les conditions observées. But: Le but de cette étude a été (1) d'analyser la microflore sous-gingivale chez les sujets DS et d'autres retardés mentaux servant de contrôles et (2) mieux connaître la microflore sous-gingivale chez les groupes de patients DS avec faible et haut risques. Matériaux et méthodes: 17 sujets DS et 17 sujets contrôles ont été analysés de manière parallèle en fonction de l'âge, du niveau de plaque dentaire et du saignement au sondage. De plus, le groupe DS était scindé en deux sous-groupes: "petit risque" (0 à 2 dents perdues pour cause de maladie parodontale; n=6), et "haut risque" (6 à 13 dents perdues; n=11). La fréquence globale et les proportions de pathogènes parodontaux putatifs l'Actinobacillus actinomycetemcomitans, le Porphyromonas gingivalis, le Prevotella intermedia, le Bacteroides forsythus, le Peptostreptococcus micros, le Fusobacterium nucleatum et le Campylobacter rectus dans la plaque sous-gingivale ont été déterminés en utilisant des techniques de culture anaérobie. Résultats: Aucune différence dans la fréquence globlale de ces bactéries ni dans la composition de la flore sous-gingivale n'a été trouvée entre le groupe DS et le groupe contrôle. Il n'y avait également aucune différence entre les sous-groupes à faible et à haut risques. Conclusions: Parce qu'aucune différence n'a été décelée dans la microflore entre les groupes DS et contrôle, aucun effet spécifique de leur flore sous-gingivale ne pourrait être responsable; les facteurs de l'hôte constituent très vraisemblablement l'explication des différences observées chez les sujets DS. [source] Equal Sex Ratios of a Marine Green Alga, Bryopsis plumosaJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 6 2008Tatsuya Togashi Abstract By finding some important culture conditions as below, we succeeded in experimentally controlling the whole life history of a dioecious marine green alga, Bryopsis plumosa (Hudson) C. Agardh. In this study, we focused on the primary and secondary sex ratios (i.e. at inception and maturity) using these culture techniques. Gametogenesis was induced by culturing haploid gametophytes with Provasoli's enriched seawater (PES) medium under a 14:10 h light : dark cycle at 14 °C. Formed zygotes grew into diploid sporophytes, which were cultured for 3 months with PES medium under a 14:10 h light : nbsp;dark cycle at 18 °C. Then they were transferred into Schreiber medium and cultured under a 10:14 h light : dark cycle at 22 °C. Within 1 week, zoosporogenesis was observed. Zoospores were released within a couple of days. Each zoospore soon germinated and grew into a unisexual gametophyte. The primary sex ratio was examined in gametophytes that originated from a single sporophyte. The secondary sex ratio was studied in the field. Both were estimated as 1:1. Synchronized meiotic cell divisions might occur during zoosporogenesis dividing each sex-determining factor evenly among zoospores. Given the equal sex ratio at maturity, there seems to be no environmental factor that differentially affects the survival of male or female gametophytes in nature. [source] Comparison of Development and Larval Growth of Four Venerid ClamsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 2 2005Young-Baek Hur The development and larval morphology of four venerid calms, Ruditapes philippinarum, Mactra veneriformis, Cyclina sinensis, and Meretrix lusoria, which cohabit the intertidal zone in western coastal Korea, were compared using laboratory culture techniques. At 87 ,m, the fertilized eggs of C. sinensis and M. lusoria were the largest and at 53 ,m, those of M. veneriformis were the smallest. D-shaped larvae of M. lusoriu were the largest and those of M. veneriformis were the smallest measuring at 135 ,m and 89 ,m, respectively. D-shaped larvae of R. philippinarum and M. lusoria had symmetrical shoulder angles and an elliptical ventral form, in contrast to the asymmetrical shoulder angles and round ventral forms of M. veneriformis and C. sinensis. In general, pediveliger larvae of all species in the study were yellow, but those of M. veneriforks and C sinenis were a more pronounced yellow. In between the early D-shaped and pediveliger stage, 7 and 17 d elapsed for M. lusonia and C. sinensis larvae, respectively. In the early larval stages for all species, the sheU length was longer than the height. However, shell length and height later became approximately the same size in all species except R. philippinarum, which exhibited a flat shape. These results indicate that for these four venerid clams, the different characteristics in larval growth and external morphology provide the evidence necessary for larval identification of natural seed production despite the fact that they spawn concurrently in the intertidal zone. [source] Microbiologic Evaluation of Gallbladder Bile of Healthy Dogs and Dogs with Iatrogenic Hypercortisolism: A Pilot StudyJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2010P.H. Kook Background: In people, hypercortisolism (HC) has been associated with acalculous cholecystitis and biliary dyskinesia, which may potentiate ascending biliary infections. In dogs, an association between HC and gallbladder disease recently has been documented, although the role of bacteria remains controversial. Furthermore, there is no information on the gallbladder bile microbial flora in healthy dogs. Objectives: To investigate the microbial flora in gallbladder bile in healthy dogs, the relationship between iatrogenic hyperadrenocorticism and bactibilia and possible changes in biliary microbial flora after cortisol withdrawal in dogs. Animals: Six control dogs and 6 dogs treated with hydrocortisone. Methods: Gallbladder bile obtained by percutaneous ultrasound-guided cholecystocentesis was cultured aerobically and anaerobically and examined cytologically before (d0), during (d28, d56, d84), and after (d28p, d56p, d84p) administration of hydrocortisone (8 mg/kg PO q12h). Results: In the control group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp.; Escherichia coli on d0) and 1/42 bile smears had cytological evidence of bacteria (d28). In the HC group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp. on d28; Bacillus sp. on d28p) and 3/42 bile smears had cytological evidence of bacteria (d84, d84, d28p). All dogs remained healthy throughout the study period (168d). Conclusions and Clinical Importance: Based on the results of conventional bacterial culture techniques, gallbladder bile of healthy dogs periodically may harbor bacteria, which do not appear to be clinically relevant. A 3-month period of iatrogenic HC was not associated with bactibilia. A higher prevalence of bactibilia may be detected with micromolecular techniques. [source] Differential binding to and biofilm formation on, HEp-2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene clusterMOLECULAR MICROBIOLOGY, Issue 5 2002Jennifer D. Boddicker Summary Type 1 fimbria-mediated adherence to HEp-2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose-sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp-2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp-2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp-2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp-2 cells using once-flow-through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process. [source] Influence of genetic background on transformation and expression of Green Fluorescent Protein in Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 5 2005W. Teughels Background/aims:, The development of an electro-transformation system and the construction of shuttle plasmids for Actinobacillus actinomycetemcomitans have enhanced the molecular analysis of virulence factors. However, inefficient transformation is frequently encountered. This study investigated the efficiency of electro-transformation and expression of Green Fluorescent Protein (GFP) in 12 different A. actinomycetemcomitans strains. The influence of the plasmid vector, serotype, and phenotype were the major factors taken into consideration. Material and methods:, Twelve serotyped A. actinomycetemcomitans strains were independently electro-transformed with two different Escherichia coli,A. actinomycetemcomitans shuttle plasmids (pVT1303 and pVT1304), both containing an identical ltx-GFPmut2 gene construct but a different backbone (pDMG4 and pPK1, respectively). The transformation efficiency, transformation frequency, and electro-transformation survival rate were determined by culture techniques. GFP expression was observed at the colony level by fluorescence microscopy. Results:, All strains could be transformed with both plasmids. However, major differences were observed for the transformation efficiency, transformation frequency, and electro-transformation survival rate between strains. The data demonstrated that plasmid vector, serotype, and phenotype are key players for obtaining a successful transformation. An inverted relationship between the electro-transformation survival rate and tranformation frequency was also observed. GFP expression was also influenced by phenotype, serotype and plasmid vector. Conclusions:, The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent. [source] Moss Systems Biology en Route: Phytohormones in Physcomitrella DevelopmentPLANT BIOLOGY, Issue 3 2006E. L. Decker Abstract: The moss Physcomitrella patens has become a powerful model system in modern plant biology. Highly standardized cell culture techniques, as well as the necessary tools for computational biology, functional genomics and proteomics have been established. Large EST collections are available and the complete moss genome will be released soon. A simple body plan and the small number of different cell types in Physcomitrella facilitate the study of developmental processes. In the filamentous juvenile moss tissue, developmental decisions rely on the differentiation of single cells. Developmental steps are controlled by distinct phytohormones and integration of environmental signals. Especially the phytohormones auxin, cytokinin, and abscisic acid have distinct effects on early moss development. In this article, we review current knowledge about phytohormone influences on early moss development in an attempt to fully unravel the complex regulatory signal transduction networks underlying the developmental decisions of single plant cells in a holistic systems biology approach. [source] Human-in-mouse modeling of primary head and neck squamous cell carcinoma,THE LARYNGOSCOPE, Issue 12 2009Jonathan H. Law MD Abstract Objectives/Hypothesis: To develop a reliable modeling system for head and neck squamous cell carcinoma (HNSCC). Study Design: Laboratory-based translational study. Methods: HNSCC tissue was obtained from patients at biopsy/resection, cultured, and implanted into mice. In vivo, tumor growth, and survival was monitored by bioluminescence imaging. Histology and immunohistochemistry (IHC) were used to confirm HNSCC and human origin. Results: Short-term culture techniques were optimized allowing survival of primary HNSCC cells more than 7 days in 76% of tumors. The size of the tumor biopsy collected did not correlate with the success of short-term culture or xenograft establishment. Xenograft modeling was attempted in primary HNSCCs from 12 patients with a success rate of 92%. Immunostaining confirmed human origin of epithelial tumor cells within the modeled tumor. Bioluminescence and Ki67 IHC suggested tumor proliferation within the model. Luciferase expression was maintained for as long as 100 days in modeled tumors. Conclusions: The techniques developed for short-term primary tumor culture followed by xenograft modeling provide a low-cost and tractable model for evaluation of HNSCC response to standard and novel therapies. The high success rate of human-in-mouse tumor formation from primary HNSCC suggests that selection pressures for tumor growth in this model may be less than those observed for establishment of cell lines. Bioluminescent imaging provides a useful tool for evaluating tumor growth and could be expanded to measure response of the modeled tumor to therapy. This model could be adapted for xenograft modeled growth of other primary tumor types. Laryngoscope, 2009 [source] Candidate's Thesis: Direct Evidence of Bacterial Biofilms in Otitis Media,THE LARYNGOSCOPE, Issue 12 2001J. Christopher Post MD Abstract Objectives/Hypothesis Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations. Study Design Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients. Methods Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes. Results Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes. Conclusions Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea. [source] Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like propertyTHE PROSTATE, Issue 12 2007Victor K. Lin Abstract INTRODUCTION The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia. Prostate 67: 1265,1276, 2007. © 2007 Wiley-Liss, Inc. [source] Developing the harpacticoid copepod Tisbe biminiensis culture: testing for salinity tolerance, ration levels, presence of sediment and density dependent analysesAQUACULTURE RESEARCH, Issue 15 2006Lília P Souza-Santos Abstract Copepods have a number of advantages for use as live food in cultures of fish and crustacean larvae. This study aimed to develop culture techniques of Tisbe biminiensis Volkmann-Rocco 1973 in volumes of 500 mL. The first experiment tested the effect of salinity on survival and fecundity. The other experiments studied the population growth comparing two levels of daily ration and the effect of sand sediment in cultures. The cultures were carried out on plastic boxes at 29°C, salinity of 34 g L,1 and 12 h light/12 h dark photoperiod with aerated filtered seawater, total renewal every other day. Adult females tolerated the decrease of salinity from 34 to 27 g L,1 but the offspring production decreased significantly. The salinity of 20 g L,1 was not tolerated at all. Tisbe biminiensis attained one of the highest rates of increase in cultures among harpacticoids (0.33 day,1) and a high density of 205 individual ind. mL,1. The carrying capacity of the population was estimated as 67 200 ind. in 500 mL recipients. In conclusion, T. biminiensis grow fast and attain high densities in cultures of 500 mL volume without sediment, feeding a daily ration of 50,100% of copepodite biomass. [source] Isolation and culture of the marine rotifer, Colurella dicentra (Gosse, 1887), from a Mississippi Gulf Coast estuaryAQUACULTURE RESEARCH, Issue 14 2006Paulinus Chigbu Abstract Few marine rotifer species (e.g. Encentrum linheii and Synchaeta cecilia) have been cultured successfully besides Brachionus plicatilis and B. rotundiformis, commonly used to rear larvae of many marine fish species. The development of culture techniques for marine rotifers smaller in size than the Brachionus species may be useful for rearing fish species for which the currently used prey are too large. We evaluated the possibility of culturing Colurella dicentra isolated from a Mississippi Gulf Coast estuary. An experiment was conducted to determine the effects of salinity (10,35 g L,1) on its population growth rate. Rotifers were fed Nannochloropsis oculata at a density of 100 000 cells mL,1 for 15 days. Colurella dicentra survived in water with a salinity of 10,47 g L,1. Densities of up to 300 rotifers mL,1 were sometimes attained in cultures. Salinity influenced C. dicentra production (P<0.001). The mean rotifer numbers at 10 g L,1 (22 840±2604 SD), 15 g L,1 (25 980±7071 SD) and 20 g L,1 (19 780±1029 SD) at the end of the experiment were similar (P>0.05), but were higher (P=0.05) than numbers at 25 g L,1 (4240±1783), 30 g L,1 (1300±264 SD) and 35 g L,1 (100±101 SD). The population growth rate (r) of the rotifers was the highest at 15 g L,1 (0.37,0.42 day,1), and the lowest at 35 g L,1 (,0.33,0.06 day,1). This is the first report of C. dicentra in the estuarine waters of the Gulf of Mexico, and also the first time it has been cultured successfully. [source] Environmental mastitis in intensive high-producing dairy herds in New South WalesAUSTRALIAN VETERINARY JOURNAL, Issue 12 2009LWC Shum Objective To determine the prevalence of mastitis pathogens in high-producing intensive dairy herds in New South Wales. Design Field survey. Procedure Milk samples from the mastitis-affected quarter were collected from cows on five high-producing dairy farms in NSW. The 820 samples were cultured using standard microbiological culture techniques. Results Bacteria or fungi were isolated from 83.3% of samples (683/820). More than two colony types were isolated from 16.7% of samples (137/820), two types from 6.6% (54/820), and one type from 52.3% (429/820). No bacteria were isolated from 24.4% (200/820) of the primary cultures, but enrichment cultures of these samples yielded single colony type bacterial isolates from 36.5% (73/200) of samples. Environmental pathogens, including coliforms, environmental Streptococcus and Staphylococcus spp., made up 91% (555/610) of isolates and accounted for 33.6% (205/610), 41.6% (254/610) and 15.7% (96/610), respectively, of isolates. Escherichia coli accounted for 76.1% (156/205) of the coliform isolates, Streptococcus uberis and Streptococcus dysgalactiae accounted for 32.3% (82/254) and 28.0% (71/254), respectively, of the environmental streptococcal isolates. Contagious pathogens were uncommon, comprising only 2.5% (15/610) of the total isolates. Conclusion The incidence and causes of mastitis are largely influenced by farm management. The relatively high prevalence of coliform mastitis in the intensive high-producing herds in this survey contrasts with the low incidence reported in surveys of pasture-based herds in Victoria. If the Australian dairy industry continues its current trend of intensification, coliform intra-mammary infections may emerge as an increasingly important cause of mastitis. [source] Extent of cell differentiation and capacity for cartilage synthesis in human adult adipose-derived stem cells: Comparison with fetal chondrocytesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Nastaran Mahmoudifar Abstract This study evaluated the extent of differentiation and cartilage biosynthetic capacity of human adult adipose-derived stem cells relative to human fetal chondrocytes. Both types of cell were seeded into nonwoven-mesh polyglycolic acid (PGA) scaffolds and cultured under dynamic conditions with and without addition of TGF-,1 and insulin. Gene expression for aggrecan and collagen type II was upregulated in the stem cells in the presence of growth factors, and key components of articular cartilage such as glycosaminoglycan (GAG) and collagen type II were synthesized in cultured tissue constructs. However, on a per cell basis and in the presence of growth factors, accumulation of GAG and collagen type II were, respectively, 3.4- and 6.1-fold lower in the stem cell cultures than in the chondrocyte cultures. Although the stem cells synthesized significantly higher levels of total collagen than the chondrocytes, only about 2.4% of this collagen was collagen type II. Relative to cultures without added growth factors, treatment of the stem cells with TGF-,1 and insulin resulted in a 59% increase in GAG synthesis, but there was no significant change in collagen production even though collagen type II gene expression was upregulated 530-fold. In contrast, in the chondrocyte cultures, synthesis of collagen type II and levels of collagen type II as a percentage of total collagen more than doubled after growth factors were applied. Although considerable progress has been achieved to develop differentiation strategies and scaffold-based culture techniques for adult mesenchymal stem cells, the extent of differentiation of human adipose-derived stem cells in this study and their capacity for cartilage synthesis fell considerably short of those of fetal chondrocytes. Biotechnol. Bioeng. 2010;107: 393,401. © 2010 Wiley Periodicals, Inc. [source] Comparison of Growth and Recombinant Protein Expression in Two Different Insect Cell Lines in Attached and Suspension CultureBIOTECHNOLOGY PROGRESS, Issue 4 2001R. A. Taticek Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPL,-Sf21-AE) and Trichoplusia ni (Tn 5,-1,4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150,160 rpm were necessary for maximum growth of suspended Tn 5,-1,4 cells compared to 125,150 rpm for Sf-21 cells. An inoculum size of 5 × 105 cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for ,-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5,-1,4 cells are 2,4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of ,-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5,-1,4 cells produced 2.6,4.4 and 2.7,3 times more ,-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the ,-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of ,-galactosidase by Tn 5,-1,4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production. [source] Experimental Escherichia coli epididymitis in rats: a model to assess the outcome of antibiotic treatmentBJU INTERNATIONAL, Issue 9 2002M. Ludwig Objective ,To assess the effect of initial antimicrobial therapy with a new highly potent quinolone (sparfloxacin) on the outcome of infection, especially acute and chronic inflammation, in a rat model of unilateral Escherichia coli epididymitis. Materials and methods ,The study included 60 Sprague-Dawley rats, each of which received 0.1 mL of an E. coli (0:6 strain) suspension (106 colony forming units/mL) injected into the right ductus deferens. At 24 h after infection an oral antimicrobial treatment with sparfloxacin was initiated in half of the animals. The rats were killed 14 days, 3 and 6 months after infection, and both epididymes and the prostate gland cultured to re-isolate E. coli. To evaluate the grade of inflammation in both epididymes, histological variables, including acute and chronic inflammation and scar formation, were evaluated and a total inflammatory score, representing the sum of all variables, computed. Results ,Whereas antimicrobial therapy eradicated the pathogen, in untreated animals the pathogen was detectable for up to 6 months after infection in the infected epididymis and/or the prostate gland, while the contralateral epididymis was sterile. The inflammatory reaction in the infected epididymis was significantly less in treated animals (P < 0.001). Subclinical nonbacterial inflammation was present in the contralateral epididymis. Conclusions ,Although adequate antimicrobial treatment eradicated the pathogen and reduced the grade of epididymal damage, inflammation was not avoided. Subclinical inflammation of the contralateral epididymis may contribute to impaired fertility. These results indicate that an inflammatory reaction initiated by bacteria might persist as a nonbacterial process despite early therapy, or by bacteria undetectable by conventional culture techniques, and may compromise male fertility. [source] Clinical and Microbiological Determinants of Ailing Dental ImplantsCLINICAL IMPLANT DENTISTRY AND RELATED RESEARCH, Issue 1 2009Giorgio Tabanella DDS ABSTRACT Background: The failure of the host tissue to establish or maintain osseointegration around dental implants is due to either occlusal or parafunctional forces, premature loading, ill-directed stress, or microbial infection. The long-term failure rate of dental implants is generally 5,10%. Although a variety of etiologies of early peri-implant bone loss (from implant placement to 1-year post-loading) have been proposed, factors associated with late implant failures are less well understood but are probably related to both the peri-implant microbial environment and host factors. Discriminating between causes of implant failure is of importance for instituting a successful implant therapy. Purpose: The objective of this cross-sectional split-mouth study was to identify clinical, radiographic, and bacterial characteristics of peri-implant disease sites. Materials and Methods: Fifteen patients with bilateral implants (Brånemark®, Nobel Biocare AB, Göteborg, Sweden; and 3iÔ implant systems, Implant Innovations Inc., Palm Beach Gardens, FL, USA) participated in the study. Sites with peri-implantitis (radiographic bone loss beyond the third implant thread) and peri-implant healthy tissues (radiographic bone level above the first implant thread) were identified in periapical radiographs using a long-cone paralleling projection technique. Microbiological identification was carried out using established anaerobic culture techniques. A descriptive statistics based on means and standard deviations was reported. Results: Peri-implant bone loss was associated with the absence of radiographic crestal lamina dura, peri-implant pocket depth, pain on chewing, and the submucosal presence of the putative periodontopathogens Tannerella forsythia, Campylobacter species, and Peptostreptococcus micros. Pain was associated with P. micros, Fusobacterium species, and Eubacterium species. Discussion and Conclusion: The absence of radiographic crestal lamina dura and the presence of suspected major periodontal pathogens seem to be associated to peri-implantitis. 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