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At least 865 records · Page 48Linked to original sources
PubMed2000

Microbiological aspects of an in situ model to study effects of antimicrobial agents on dental plaque ecology.

This study validates an in situ model for ecological studies of dental plaque exposed to various antimicrobial agents with different modes of action on plaque bacteria. Eleven subjects wore two acrylic appliances, each containing two bovine enamel discs, during two 1-wk test periods. Using a split-mouth crossover design, the appliances were dipped twice daily for 1 min into water (control; treatment A), fluoride (26.3 mM NaF; B), zinc acetate (20.0 mM; C), or fluoride plus zinc acetate (D). Four of the subjects used also chlorhexidine diacetate (2.2 mM; E) and chlorhexidine plus fluoride (F). At the end of each period, plaque was collected from the discs, after which the microbiota were analyzed by culture, automated quantitative immunofluorescence, and a viability fluorescence stain. As compared to control, treatments B, C, and D resulted in a significant reduction of individual taxa as detected by immunofluorescence, whereas similar bacterial viability and total bacterial numbers were observed. In contrast, chlorhexidine significantly reduced bacterial viability, total cell numbers, and the abundance of most of the enumerated taxa. We conclude that this in situ model is well suited to study effects of antimicrobial agents on dental plaque ecology. Combined with viability testing, immunofluorescence is obviously superior to culture in detecting taxa-specific shifts caused by antimicrobial agents.

Adult↗
PubMed2000

Effect of antibiotics on viability staining of Escherichia coli in solid phase cytometry.

Solid phase cytometry (SPC) has been investigated as a tool to assess the effect of antibiotics on the viability of Escherichia coli. After exposure of the cells to the antibiotic, they are retained on a polyester membrane filter and labelled using a fluorescein derivative as a substrate for intracellular esterases. The number of fluorescent bacteria is automatically counted in an Ar laser scanning device. In the presence of nutrients, all antibiotics tested in concentrations exceeding the MIC inhibited the multiplication of cells but not the labelling per se. However, when no nutrients were added, the cells did not multiply, and inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at sub-MIC concentrations. The selective detection by SPC of membrane-permeabilizing antibiotics corroborates the requirement of membrane integrity for viability labelling of bacteria. This selectivity has been exploited to develop a method for the detection of colistin residues in milk.

Anti-Bacterial Agents↗
PubMed1989

Studies on mixed populations of human intestinal bacteria grown in single-stage and multistage continuous culture systems.

Mixed intestinal bacteria were grown for 336 h in two identical single-stage chemostats at low growth rates in a carbohydrate-limited medium. Complex bacterial populations were maintained and anaerobes always outnumbered aerobes. The predominant organisms belonged to the genera Bacteroides, Bifidobacterium, Lactobacillus, Clostridium, Eubacterium, Propionbacterium, Peptococcus, and Peptostreptococcus. Bacteroides species predominated in both fermentors, particularly B. ovatus and B. thetaiotaomicron. A high degree of reproducibility of bacteriological and fermentation product data was obtained in these experiments. When gut contents were inoculated into a five-stage continuous culture system (retention time of 79 or 38 h) containing soya bran, the medium flow rate had little quantitative effect on the formation of acidic fermentation products; however, more oxidized fermentation acids were produced at the higher retention time. Diverse bacterial populations were maintained in every vessel at each flow rate. Bacteroides fragilis group organisms, especially B. ovatus, were numerically the most important. The viability of bacteria decreased through the system, especially at a retention time of 79 h, when the bacteria were growing under severely nutrient-limited conditions.

Bacteria↗
PubMed1999

Spent culture supernatant of Mycobacterium tuberculosis H37Ra improves viability of aged cultures of this strain and allows small inocula to initiate growth.

Spent culture supernatant from early-stationary-phase Mycobacterium tuberculosis H37Ra cultures increased the viability of bacilli from aged cultures of this strain and allowed small inocula to initiate growth in liquid culture. The resuscitation factor was acid labile and heat stable, with a mass of less than 1,375 Da.

Colony Count, Microbial↗
PubMedDate not supplied

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.

The number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2-3 mm3) and with volumes tissue ratios > or =2 ml/g tissue. For incubations < or =10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions < or =10 h (collagenase 2700-3900 Mandl U/ml, neutral protease 5100-10,000 caseinase U/ml, and clostripain 35-48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350- 3000 U/ml, neutral protease 2550-7700 U/ml, and clostripain 18-36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1 x 10(4) cells/cm2. These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.

Fibroblasts↗
PubMed2004

Viability of milk neutrophils and severity of bovine coliform mastitis.

To study the host-pathogen interactions during Escherichia coli mastitis, we first determined whether E. coli infection would change blood and milk polymorphonuclear neutrophil (PMN) chemiluminescence (CL) and viability. We then hypothesized that when E. coli invade the mammary gland, the viable PMN in milk would efficiently phagocytose and destroy E. coli before establishment of infection. We observed that the phagocytosis-dependent and independent CL were closely linked to PMN viability and were crucial to the outcome of mastitis. Maximal PMN influx and colony-forming units in infected quarters appeared at postinfection hours (PIH) 6 to 24. This further boosted PMN recruitment through bone marrow-blood barrier as well as blood-milk barrier. The survival of recruited PMN in the E. coli-infected quarters was much higher than that of noninfected quarters. Chemiluminescence activity of PMN from the infected quarters significantly increased following E. coli infection, even exceeding that of blood at PIH 6, 12, and 18 to 24; no such increase was observed in noninfected quarters, suggesting that the various responses of milk PMN to stimuli resulted largely from PMN viability. The highest CL intensity and durability was observed in milk PMN from infected quarters at PIH 12. Whereas an increased viability of PMN in the noninfected quarters was only significant at PIH 6, the viability of PMN in infected quarters was long lasting and significantly higher at PIH 6 to 72. Importantly, higher preinfection milk PMN viability correlated with bacterial clearance, which was accompanied by faster recovery. Our study strongly supports the hypothesis that boosting milk PMN viability could be a strategy with which to prevent or reduce the severity of coliform mastitis in dairy cows. This strategy might be achieved through strengthening bone marrow functionality.

Animals↗
PubMed1997

Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors.

In the first of two experiments investigating the effect of dietary urea on the survival and metabolism of ovine embryos, 30 Border Leicester x Scottish Blackface ewes received a maintenance diet (milled hay, molasses, minerals, vitamins) with no urea (control, C; n = 10) or with added urea at 15 g (low urea, LU; n = 10) or 30 g (high urea, HU; n = 10) kg-1 feed for a 12 week period. The degraded nitrogen (N) status relative to estimated rumen microbial N requirements was -2, +9 and +20 g per day, respectively. One week after allocation to diets, progesterone priming (12 days) commenced. Ewes received 800 IU of equine chorionic gonadotrophin at progesterone withdrawal, were inseminated 52 h later (Day 0) and embryos were collected from five ewes per group at Day 4 and from five ewes at Day 11. If available, one embryo was returned to each ewe; the rest were cultured in vitro. There was no effect of treatment on progesterone, luteinizing hormone (LH), or time of oestrus onset C, LU and HU plasma urea (P < 0.001) and ammonia levels (C vs. HU, P < 0.01; LU vs. HU, P < 0.05) differed. Day 4 HU embryos were retarded relative to C and LU embryos. After 3 days of culture, 70%, 66% and 0% of C, LU and HU embryos, respectively, were viable. Mid-term pregnancy rates following transfer were 63%, 43% and 33%. Only one HU lamb (male) was born following embryo transfer, its birthweight (10.1 kg) exceeded that of its C (n = 3; 7.0, 7.0, 7.5 kg) and LU (n = 2; 7.3, 8.2 kg) counterparts (P < 0.025). In the second experiment, C2 (2.5 g urea kg-1; n = 5) and HU2 (30 g kg-1; n = 7) diets which provided similar intakes of degraded N relative to microbial requirements as those for C and HU ewes in Experiment 1 were fed to Border Leicester x Scottish Blackface ewes superovulated with 16 mg of porcine follicle-stimulating hormone. Urea and ammonia levels in utero-oviductal samples were elevated in HU2 ewes (P < 0.05). At collection (Day 3), HU2 embryos used more glucose (P < 0.01) and, following culture, some exhibited up to a 2.8-fold increase in metabolism. In conclusion, excess rumen degradable N in ewe diets elevates urea and ammonia in plasma and in utero, with an associated increase in embryo mortality. Nevertheless, metabolism appears to be up-regulated in some embryos and, among those that survive, fetal growth appears to be enhanced.

Ammonia↗
PubMed1977

Dependence of cytoplasmic on mitochondrial protein synthesis in K. lactis CBS 2360. II. Genetic studies.

A few eryR mutants independently isolated from K. lactis CBS 2360 display a conditional lethal phenotype at the temperature of 36 degrees C. In addition to drug resistance, also conditional lethality shows a non-Mendelian pattern of inheritance and is affected by exposure of the cells to Ethidium Bromide, indicating that in this yeast mitochondrial DNA controls cell viability. The results obtained from biochemical analysis suggest that the cellular functions in which are involved the gene products of the mitochondrial mutants analized are cytoplasmic protein and RNA syntheses.

Cytoplasm↗
PubMed2006

Development of alternative support system for viable count of cyanobacteria by most probable number method.

A technique was developed to evaluate alternative support systems to test tubes used in the standard most probable number technique, for simultaneous isolation and enumeration of cyanobacteria. Five different support systems were tested for their suitability in terms of accuracy, sensitivity, economics and ease of handling. PCR plates with 96 wells and carrying capacity of 300 microL per well were found to be most sensitive, besides being cost- and time-effective. This technique can also be useful for isolation of cyanobacteria, due to immobilization of colonies in the gel matrix and storage of samples at room temperature, without loss of viability for 5-6 weeks. This technique can help to process large sample size with ease--both for enumeration and isolation and can be extended for enumeration of other microorganisms from diverse sources.

Colony Count, Microbial↗
PubMed1990

Absence of neural crest cells from the region surrounding implanted notochords in situ.

Avian neural crest cells migrating along the trunk ventral pathway are distributed throughout the rostral half of the sclerotome with the exception of a neural crest cell-free space of approximately 85 microns width surrounding the notochord. To determine if this neural crest cell-free space results from the notochord inhibiting neural crest cell migration, a length of quail notochord was implanted lateral to the neural tube along the neural crest ventral migratory pathway of 2-day chicken embryos. The subsequent distribution of neural crest cells was analyzed in embryos fixed 2 days after grafting. When the donor notochord was isolated using collagenase, neural crest cells avoided the ectopic notochord and were absent from the area immediately surrounding the implant (mean distance of 43 microns). The neural crest cell-free space was significantly less when notochords were isolated using trypsin or chondroitinase digestion and was completely eliminated when notochords were fixed with paraformaldehyde or methanol prior to implantation. The implanted notochords did not appear to affect the overall number of neural crest cells, and therefore were unlikely to exert this effect by altering their viability. These results suggest that the notochord produces a substance that can inhibit neural crest cell migration and that this substance is trypsin and chondroitinase labile.

Animals↗
PubMed2001

In vitro activity of the chelating agents nitroxoline and oxine against Mycobacterium bovis BCG.

The chelating antibiotic nitroxoline (5-nitro-8-hydroxyquinoline) showed a bacteriostatic effect at a concentration of 10 microM for Mycobacterium bovis BCG. At higher concentrations the compound showed moderate cidal activity against growing bacilli. In contrast, its non-nitrated derivative oxine (8-hydroxyquinoline, MIC=2 microM) reduced the viability of a growing culture rapidly, 5000-fold at a concentration where the nitroxoline was merely bacteriostatic. Both compounds showed appreciable cidal activity against bacilli in their hypoxic dormant state.

Animals↗
PubMed2003

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Amino Acid Sequence↗
PubMed2005

Survival of Enterococcus faecalis in root canals ex vivo.

AIM: The hypotheses tested in this study were that: (i) Enterococcus faecalis can survive long-term entombment in root filled teeth without additional nutrients, (ii) initial cell density influences the survival of E. faecalis in instrumented root canals and (iii) gelatinase-production capacity influences the survival of E. faecalis in root canals. METHODOLOGY: The root canals of 150 extracted single canal teeth were instrumented to apical size 60 and divided into six groups of 25. Within each group 10 canals were inoculated with either gelatinase-producing E. faecalis OG1-S and the other 10 with its gelatinase-defective mutant E. faecalis OG1-X. Five canals per group were kept as uninoculated controls. The root canals in groups 1 and 2 were inoculated with 10(6) bacteria, incubated for 48 h at 37 degrees C then filled with gutta-percha and zinc-oxide eugenol sealer. Root canals were inoculated with 10(6), 10(5), 10(4) and 10(3) bacteria in groups 3-6, respectively, and left unfilled. All teeth were sealed coronally with glass-ionomer cement. After 6- (groups 1, 3-6) and 12-month (group 2) incubation at 37 degrees C in 100% humidity, root fragments were analysed for presence of E. faecalis, using culture, polymerase chain reaction and histological methods. RESULTS: Viable E. faecalis was recovered from all root filled teeth and from 95-100% of unfilled inoculated teeth. Initial cell density and gelatinase production did not influence the recovery of viable E. faecalis (P > 0.05; chi-square test). Enterococcus faecalis 16S rRNA gene products were present in all inoculated teeth and absent in all noninoculated controls. Dentinal tubule infection was evident under light microscopy in sections from inoculated teeth after 48-h, 6- and 12-month incubation. CONCLUSIONS: Enterococcus faecalis inoculated into root canals maintained viability for 12-months ex vivo. The clinical implications are that viable E. faecalis entombed at the time of root filling could provide a long-term nidus for subsequent infection.

Adaptation, Physiological↗
PubMed2004

Use of a fluorescent viability stain to assess lethal and sublethal injury in food-borne bacteria exposed to high-intensity pulsed electric fields.

AIMS: To apply scanning electron microscopy, image analysis and a fluorescent viability stain to assess lethal and sublethal in food-borne bacteria exposed to high-intensity pulsed electric fields (PEF). METHODS AND RESULTS: A rapid cellular staining method using the fluorescent redox probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 4',6-diamidino-2-phylindole was used for enumerating actively respiring cells of Listeria mononcytogenes, Bacillus cereus and Escherichia coli. This respiratory staining (RS) approach provided good agreement with the conventional plate count agar method for enumerating untreated and high-intensity PEF-treated bacteria suspended in 0.1% (w/v) peptone water. However, test organisms subjected to similar levels of lethality by heating at 56 degrees C resulted in ca 3-log-unit difference in surviving cell numbers ml(-1) when enumerated by these different viability indicators. PEF-treated bacteria were markedly altered at the cellular level when examined by scanning electron microscopy. CONCLUSIONS: While PEF-treatment did not produce sublethally injured cells (P < 0.05), substantial subpopulations of test bacteria rendered incapable of forming colonies by heating may remain metabolically active. SIGNIFICANCE AND IMPACT OF THE STUDY: The fluorescent staining method offers interesting perspectives on assessing established and novel microbial inactivation methods. Use of this approach may also provide a better understanding of the mechanisms involved in microbial inactivation induced by PEF.

Bacillus cereus↗
PubMed2000

Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460.

The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

ATP-Binding Cassette Transporters↗
PubMed2001

Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes.

To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.

Adaptation, Physiological↗
PubMed1986

Phenotypic and cytotoxic characteristics of the immune cells of the human placenta.

Despite some functional impairment of the newborn's T-cell immune system, most infants survive the intrauterine and perinatal period without succumbing to infection or maternal lymphocyte engraftment. The placenta may play a crucial role in protecting the infant from microbial and histocompatibility antigens. Accordingly, we studied phenotypic and functional capacities of placental cells. Placentas were obtained from uncomplicated pregnancies. Matched cord blood and maternal peripheral blood were also obtained in many instances. Fresh minced placental tissue was washed and digested with collagenase and DNase and mononuclear cells were obtained by density gradient centrifugation. The average yield was 10(6) cells/g of tissue with greater than 80% viability. Chromosome analysis of five placental preparations indicated that these cells were of fetal rather than maternal origin. The isolated placental cells consisted of trophoblasts, lymphocytes (74 +/- 3%), monocytes (16 +/- 3%), and granulocytes (8 +/- 2%). E-rosette forming cells (T cells) made up 65 +/- 2% and surface membrane immunoglobulin positive cells made up 8 +/- 1% of the placental mononuclear cells. Fluorescent activated analysis of the mononuclear cells indicated less Leu 4-positive cells (Pan-T) 43 +/- 3%, and less Leu 3-positive (T-helper cells) (25 +/- 2%), than cord and maternal cell preparations. Leu-2, DR, and B1 positive cells were similar to those in cord and maternal blood. Leu 7 and especially Leu 11 positive cells, markers for natural killer cells, were abundant in placental cells, making up 4 +/- 0.7% and 20 +/- 3%, respectively. The Leu 7/Leu 11 ratio of the placental cells was different from either the maternal or cord blood cells. Natural killer activity of placental cells against a K562 natural killer target was low, despite the abundance of cells with NK markers. The K562 activity was low in the placental cells, similar to the low NK activity of maternal and cord cells. Molt 4f killer activity was near normal. Lectin-dependent cytotoxicity using an EL-4 cell target plus PHA was low in placentas, compared to normal, maternal, or cord cell cytotoxicity. Matched samples indicated that LDCC activity was mother greater than cord greater than placenta. Antibody-dependent cytotoxicity (Raji target) of placental cells showed low activity, and again the paired studies indicated that normal controls greater than maternal greater than cord greater than placenta cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗