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PubMed1983

Harvesting and subculturing cells growing on denatured-collagen coated microcarriers (Cytodex 3).

Selecting correct procedures and conditions for harvesting cells was essential for successful subcultivation of cells when using the microcarrier culture technique. The proteolytic enzymes trypsin, Dispase and collagenase were compared with respect to the yield and the viability of Vero cells when harvested from Cytodex 3 microcarriers. Treatment with Dispase or trypsin resulted in similar viabilities but recovery after trypsin treatment was improved. The advantage of using collagenase was the improved plating efficiency of cells when subculturing to new microcarrier cultures. Pre-washing the confluent microcarriers with EDTA (0.02% w/v) improved the yield and the viability when harvesting with trypsin or collagenase. The stage of the culture cycle when cells were harvested did not influence relative recovery or viability. In contrast, significant differences in recovery and viability were found when harvesting cells grown in the presence of different batches of foetal calf serum. The results confirm that the choice of enzyme for harvesting depends upon the cell type and purpose of the culture.

Animals↗

PubMed1995

Drug-resistant epimastigotes of Trypanosoma cruzi and persistence of this phenotype after differentiation into amastigotes.

In vitro benznidazole resistance was induced in cloned T. cruzi epimastigotes using a continuous drug pressure protocol. Stocks were selected according to their previous genetic characterization by multilocus enzyme electrophoresis. All the resistant clones were able to grow in long term cultivation in the presence of at least 50 microM benznidazole, which is the drug plasma level during chemotherapy in man. The highest level of resistance achieved was 220 microM. After differentiation of the epimastigote into the amastigote forms, the drug-resistance level was not affected. In both, the resistant epimastigotes and the resistant amastigotes, growth curves exhibited a lower growth rate than the sensitive counterparts without affecting the viability of the parasites. These data could be significant in basic research, to study the drug-resistance phenotype on relevant chemoresistant clones of T. cruzi, and to follow this phenotype after in vivo cycles.

Animals↗

PubMed2004

Morphological and physiological changes in Saccharomyces cerevisiae by oxidative stress from hyperbaric air.

Increase in air or oxygen pressure in microbial cell cultures can cause oxidative stress and consequently affect cell physiology and morphology. The behaviour of Saccharomyces cerevisiae grown under hyperbaric atmospheres of air and pure oxygen was studied. A limit of 1.0 MPa for the air pressure increase (i.e. 0.21 MPa of oxygen partial pressure) in a fed-batch culture of S. cerevisiae was established. Values of 1.5 MPa air pressure and 0.32 MPa pure oxygen pressure strongly inhibited the metabolic activity and the viability of the cells. Also, morphological changes were observed, especially cell-size distribution and the genealogical age profile. Pressure caused cell compression and an increase in number of aged cells. These effects were attributed to oxygen toxicity since similar results were obtained using air or oxygen, if oxygen partial pressure was equal to or higher than 0.32 MPa. The activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD) (cytosolic and mitochondrial isoformes) indicated that the enzymes have different roles in oxidative stress cell protection, depending on other factors that affect the cell physiological state.

Adaptation, Physiological↗

PubMed2005

Down-modulation of nitric oxide production in murine macrophages treated with crude plant extracts from the Brazilian Cerrado.

Several plant species from the Cerrado biome in Brazil are popularly used as herbal medicines for its reputed analgesic, anti-acid, anti-microbial, anti-inflammatory and anti-tumoral properties, among others. It has been reported that some plant extracts interfere in the production of nitric oxide (NO), an important inflammatory mediator. In the present study, we investigated the effect of hexanic and ethanolic extracts from three plant species on NO production by LPS/IFN-gamma-activated J774 macrophages based on traditional use. The cytotoxic effect of the crude extracts was determined by the thiazolyl blue test (MTT) to measure cell viability. Serjania lethalis stem extracts and Cupania vernalis leaf extracts significantly inhibited NO production, while extracts from Casearia sylvestris var. lingua were inactive or showed low activity on NO production, or were very cytotoxic. The ethanolic stem bark and leaf extracts of Serjania lethalis and Cupania vernalis, respectively, almost completely inhibited the production of NO by J774 macrophages. It can be concluded that the selected extracts are potential sources of active compounds that might be used as anti-inflammatory agents.

Animals↗

PubMed2001

Toxic effects of linear alkylbenzene sulfonate on metabolic activity, growth rate, and microcolony formation of Nitrosomonas and Nitrosospira strains.

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC(50)], 6 to 38 mg liter(-1)) was affected much less by LAS than the growth rate and viability (EC(50), 3 to 14 mg liter(-1)) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter(-1)); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH(4)(+) oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.

Alkanesulfonic Acids↗

PubMed2005

Validation of respirometry as a short-term method to assess the efficacy of biocides.

This study shows that a short-term respirometric measurement based on the rate of oxygen uptake needed to oxidize glucose is a reliable and fast method to assess biocide efficacy against P. fluorescens cells. Respiratory activity using oxygen consumption rate, the determination of viable and nonviable cells using Live/Dead BacLight kit and colony formation units (CFU), were compared as indicators of the biocidal efficacy of ortho-phthalaldehyde (OPA). The results showed that determining the effect of OPA against P. fluorescens using the different methods leads to different conclusions. The minimum bactericidal concentration (MBC) was 80 mgl(-1), 100 mgl(-1) and 65 mgl(-1) respectively, using respiratory activity, viability using BacLight counts and culturability. The plate count method was shown to underestimate the biocidal action of OPA, whilst data from respirometry and viability using Live/Dead BacLight kit correlated strongly and were not statistically different when yellow cells were considered nonviable. Respirometry therefore represents an expeditious, non-destructive and accurate method to determine the antimicrobial action of biocides against aerobic heterotrophic bacteria.

Bacteriological Techniques↗

PubMed1997

Structural analyses of plaque and caries in relation to the morphology of the groove-fossa system on erupting mandibular third molars.

Using 22 erupting human third mandibular molars, this study investigated: (1) the relationship between the morphology of the interlobal groove and the histological features of caries, (2) the ultrastructural features of the interlobal groove contents, and (3) whether the morphology of interlobal grooves influences the viability of the microorganisms. Posteruption time was classified as (1) < 3 months (7 teeth), (2) 3-12 months (8 teeth) and (3) > 12 months but < 18 months (7 teeth). The teeth were pre- and postfixed, and buccolingual sections were prepared. The mesial/distal interlobal groove on 116 sections was classified as fissure-like or groove-like. All sections were embedded and ground, and microradiographs were made. The interlobal groove contents were analyzed on two sections from each tooth, one with a fissure-like and one with a groove-like morphology, giving a total of 44 sections. Eighteen teeth (82%) had evidence of caries. Severity of caries was associated with length of posteruption time. 'Fissures' were not more prone to caries than 'grooves'. In about 70% of the 'fissures' caries had penetrated deepest at the entrance rather than in the deeper parts. LM and TEM examinations from 1 tooth disclosed erythrocytes, enamel organ cells and pioneers of microorganisms in the interlobal grooves. All other interlobal grooves were occupied by microorganisms, mainly gram-positive coccoids and small rods. Viable microorganisms were seen at the entrance as well as at the bottom part of the 'grooves'. In 'fissures' viable microorganisms were primarily observed at the entrance, while in deeper parts the microorganisms were less viable or dead. A moderate association was observed between the viability of the microorganisms and differences in the depths of caries penetration in the fissures. Thus, the internal morphology of the interlobal grooves influenced the conditions for bacterial growth and this determined the location for caries progression within the groove-fossa system.

Adolescent↗

PubMed1989

Fate of Staphylococcus aureus in whey, whey cream, and whey cream butter.

Fresh Cheddar cheese whey was inoculated with ca. 10(6) Staphylococcus aureus/ml and held at 4, 25, and 37 degrees C for 48 h. Numbers of staphylococci decreased in whey at 25 and 37 degrees C and decreased or remained constant in whey at 4 degrees C. When Cheddar cheese whey was neutralized with sodium hydroxide before inoculation with ca. 10(2) or 10(6) S. aureus/ml, numbers of the bacterium increased at all incubation temperatures. Viability of S. aureus strains in whey butter made from inoculated whey cream (from Cheddar cheese whey) was determined. Whey cream was either neutralized to a titratable acidity of .15% or untreated before inoculation with ca. 10(4) S. aureus/ml. Butter churned from the whey cream was held at 4, 25, and 30 degrees C for up to 4 wk. Viability of S. aureus was enhanced in lightly salted (1%) whey cream butter and in butter made from neutralized whey cream. Strains of S. aureus did not survive in unsalted or in salted (1.5%) butter made from untreated whey cream.

Animals↗

PubMed1980

Effect of sodium chloride on gentamicin accumulation by Escherichia coli: correlation with bacterial growth and viability.

The kinetics of gentamicin accumulation by a sensitive strain of Escherichia coli were investigated at gentamicin concentrations from 0.02 to 200 microgram/ml. Accumulation with time shows two energy-dependent phases and is saturable. Sodium chloride delays the onset of the second more rapid energy-dependent phase and decreases the magnitude of gentamicin accumulation for incubations up to 60 minutes at all gentamicin concentrations tested. Simultaneous determinations of accumulation, cell viability, and growth inhibition indicate that antimicrobial activity is correlated with the magnitude of gentamicin accumulation. These observations suggest that altered bacterial accumulation of gentamicin explains the effect of sodium chloride on the antimicrobial activity of gentamicin.

Escherichia coli↗

PubMed2003

Direct estimate of active bacteria: CTC use and limitations.

During the last 10 years, the dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been used to determine the in situ number of "active" bacteria in different ecosystems. A part of this success is due to a simple protocol, which does not require sophisticated equipment. However, it has not been established whether the method determines viable cells, e.g. those capable of growth and cell division, as opposed to cells that are active in the sense of having some detectable metabolic activity. In this study, the number of CTC-positive cells through the growth stages of Escherichia coli was estimated and compared to counts of the total number of bacteria, the culturability (CFU counts) and respiratory activity (CO(2) evolution). There was a good correlation between the number of CTC-positive cells and the CFU count, regardless of the growth phase. However, CTC could still be reduced by a large part of the population during the first hours of stationary phase even if the bacteria were no longer releasing CO(2). Thus, the reduction of CTC is a good estimator for cell viability, rather than cell activity. Additionally, a review of the literature showed that there is presently no standardized protocol for using CTC, which makes difficult at present the comparison of active bacterial numbers in different samples from different sites.

Bacteriological Techniques↗

PubMedDate not supplied

Effects of dexamethasone and FK506 on Helicobacter pylori-induced gastritis and bacterial viability in Mongolian gerbils.

FK506 and dexamethasone were used to investigate whether or not immunosuppression affects H. pylori colonization and gastric mucosal damage induced by Helicobacter pylori in Mongolian gerbils. Two weeks after H. pylori infection, FK506 and dexamethasone or vehicle alone were subcutaneously administered once daily for the following 2 weeks. FK506 or vehicle alone was administered subcutaneously once daily for 5 weeks (1 week before and 4 weeks after infection). In H. pylori-infected animals for 4 weeks, hemorrhagic erosions and inflammatory responses (neutrophil infiltration and lymphoid follicle formation) were induced in gastric mucosa at an incidence of 100%. Both FK506 and dexamethasone administered for 2 weeks markedly reduced such mucosal changes. In these animals, H. pylori viability in the stomach was significantly elevated. FK506 administered for 5 weeks also significantly inhibited the hemorrhagic erosions, edema and neutrophil infiltration in the stomach. H. pylori viability was slightly elevated as compared with the control. It was concluded that the host immune responses might play dual roles both by deteriorating gastritis induced by H. pylori and by protecting against H. pylori infection in its early stage.

Animals↗

PubMed1982

Rapid transient growth at low pH in the cyanobacterium Synechococcus sp.

The thermophilic cyanobacterium Synechococcus sp. strain Y-7c-s grows at its maximum rate at a high pH (pH 8 and above) the does not show sustained growth below pH 6.5. However, rapidly growing, exponential-phase cells from high-pH cultures continued to grow rapidly for several hours after transfer to pH 6.0 or 5.0. This transient growth represented increases in mass and protein, but cells failed to complete division. Viability loss commenced well before the cessation of growth, and cells at pH 5.0 showed no net DNA synthesis. When irradiated by visible light, cells at pH 6.0 and 5.0 maintained and internal pH of 6.9 to 7.1 (determined by 31P nuclear magnetic resonance spectroscopy) and an extremely high ATP/(ATP + ADP) ratio even after growth had ceased. Cells exposed to a low pH did not show an increase in the spontaneous mutation rate, as measured by mutation to streptomycin resistance. However, cells already resistant to streptomycin were more resistant to viability loss at a low pH than the parental type. Cultures that could grow transiently at a low pH had higher rates of viability loss than nongrowing cultures in light or darkness. The retention of a high internal pH by cells exposed to a low pH suggested that a low pH acted initially on the cell membrane, possibly on solute transport.

Adenosine Diphosphate↗

PubMed1985

High-yield preparation of porcine hepatocytes for long survival after transplantation in the spleen.

The creation of an auxiliary liver by autotransplantation of liver parenchymal cells into the spleen has mainly been studied in rats for the treatment of acute liver failure. In order to apply this procedure to humans with chronic liver insufficiency the aim of this work was: To demonstrate that hepatocytes can survive for long periods after autotransplantation into the spleen; to increase the yield of the isolation of hepatocytes obtained from pig livers since this animal has a more fibrous liver than rats or normal humans and consequently one which is more difficult to dissociate. In 21 pigs isolated hepatocytes were obtained with in collagenase dissociation technique, the yield being 1-3 X 10(7) cells per gram of liver and the viability 70-95%. The hepatocytes survived and maintained normal morphological and histochemical characteristics up to 7 months after transplantation, the date of sacrifice of the last animal.

Animals↗

PubMed1991

Adhesion of Helicobacter pylori to human gastric mucosal biopsy specimens cultivated in vitro.

The therapeutic advances in Helicobacter pylori infection is hampered by the lack of suitable animal model systems. We have previously reported on successful adherence of H. pylori to pig gastric mucosa cultured in vitro. The aim of this study was to verify the technique in human biopsy specimens cultured in vitro. Mucosal samples were taken from H. pylori-negative and H. pylori-positive patients undergoing gastric surgery. The non-infected tissue was infected with H. pylori in vitro, and the infected tissue was put into culture immediately. Total number and those H. pylori firmly attached were checked throughout a 72-h culture. Viability of cultured human gastric mucosa was good and unaffected by the presence of H. pylori. The amount of bacteria adhering, increased with time from 0.01% to 2-4% after 72 h in culture. In vivo-infected specimens initially had a low number of firmly attached H. pylori, but total H. pylori increased with time in culture. It is concluded that human gastric biopsy specimens show good viability for 72 h and that viability and cell division of H. pylori were maintained in both in vivo and in vitro H. pylori-infected tissue. In both cases the total number of viable bacteria attached to the specimens increased with incubation time.

Animals↗

PubMed2002

Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure.

A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.

Anti-Bacterial Agents↗

PubMed1993

Antimicrobial activity of sulfur dioxide to certain lactic acid bacteria isolated from wines.

This study was conducted to investigate the relationship between pH and the toxicity of sulfur dioxide against homo- and hetero-lactic acid bacteria isolated from American acidic wines. Malolactic fermentation was found to be growth-associated in homo- and hetero-lactic strains. The pH of CBB broth had an insignificant effect on the specific death rate of these strains; the concentration of molecular sulfur dioxide had a great effect on the specific death rate and malate degradation. Laboratory or freshly isolated strains were exposed to various concentrations of free sulfur dioxide at pH 3.8 and 3.4. At pH 3.4, sulfur dioxide was more effective against lactic acid bacteria than at pH 3.8 since more was present as molecular sulfur dioxide and causing an increase in the specific death rate. Pediococcus strain BB was more resistant to sulfur dioxide than those hetero-lactic fermentation strains such as Leuconostoc oenos PSU-1 and ML-34. The effect of sulfur dioxide concentration on the viability of L. oenes ML-34, PSU-1, and Pediococcus strain BB appeared to be biphasic. No or little L-malic acid was degraded when the homo- and hetero-fermentative strains were killed by sulfur dioxide.

Hydrogen-Ion Concentration↗

PubMed2001

Genomics-based identification of targets in pathogenic bacteria for potential therapeutic and diagnostic use.

The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.

Amino Acyl-tRNA Synthetases↗

PubMed2007

Anethole, a potential antimicrobial synergist, converts a fungistatic dodecanol to a fungicidal agent.

Anethole shows synergistic effects on the antifungal activities of phytochemicals including polygodial and (2E)-undecenal against Saccharomyces cerevisiae and Candida albicans. It was found that a fungistatic dodecanol combined with a sublethal amount of anethole showed a fungicidal activity against S. cerevisiae. The MIC of dodecanol quickly reduced cell viability, but the cell viability recovered shortly after and then finally became no longer different from the control, indicating that the effect of dodecanol on this yeast was classified as sublethal damage. On the other hand, anethole completely restricted the recovery of cell viability. Therefore the expression of the synergistic effect was probably due to a blockade of the recovery process from dodecanol-induced stress.

Allylbenzene Derivatives↗