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A modified version of the two-step enzymatic method for isolation of hepatocytes from the liver of adult mice is described. The method yields 6.10(7)-7.10(7) cells with viability of 70-80%. It is simple and economical, requiring no great amounts of collagenase.
The effects of cross-linking agents on the resistance of the articular surface to digestion with clostridial collagenase were studied using a described in vitro system. Mouse femoral heads were treated with various concentrations of glutaraldehyde, with osmium tetraoxide and with dithiobis (succinimydil propionate), digested with the enzyme, labeled with cationized ferritin and examined by electron microscopy. Collagenase alone caused disruption of the articular surface with penetration of the large marker into the cartilage matrix. After treatment of the femoral heads with the cross-linking agents, no effects on the morphology and on the labeling of the articular surface and no penetration of the label into the cartilage matrix were observed. Increasing cross-linking at the articular surface might be a new route for therapeutic intervention. However, experiments would be needed to assess the effect of the procedure on the viability and nutrition of chondrocytes and on the functional properties of the tissue.
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The Institut Gustave-Roussy (IGR) Department of Clinical Pharmacy (DCP) ensures the annual preparation of about 30 000 therapeutic batches of anti-neoplastic agents. High performance thin-layer chromatography (HPTLC) allows postproduction quality control of these batches. Although the centralized chemotherapy manufacturing unit has been recently ISO 9001:2000 certified, it was considered to improve the quality level of manufactured batches even further. The viability of micro-organisms (bacteria and fungi) in appropriate sterile media containing various anti-neoplastic agents at therapeutic concentration was assessed to demonstrate the lack of contamination during our manufacturing process in the isolator. After 14 days of incubation in these media, the results show the absence of contamination of the manufactured batches. This leads us to conclude that using sterile drugs and sterile medical devices in a sterile isolator allows the manufacture of sterile therapeutic batches with excellent confidence.
Comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied on Zymomonas mobilis cultures, was performed. The slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated the Zymomonas cell viability. The slide technique is advantageous also because it gives information on the cell morphology changes, notably the abnormal cell elongation, in the ethanol fermentation.
Physical treatments, like heating or irradiation, may reduce the viability or eradicate Aspergillus conidia, which in turn might help to prevent infections by members of this genus. Chemical treatments can also prevent infection resulting from contaminated hospital fabrics or surfaces. Our objectives were to study the kinetics of survival of the conidia of pathogenic Aspergillus species, like A. fumigatus, A. flavus and A. niger, during exposure to heating at 60 degrees C and microwave irradiation. In addition, we evaluated the susceptibility patterns of Aspergillus conidia to such chemical agents as cupric sulphate and sodium hypochlorite. Heating the conidia of A. flavus and A. niger at 60 degrees C for 45 min was found to be fungicidal (reduction > 104 conidia/ml), but was not with A. fumigatus conidia. Short periods of microwave irradiation (40 s) resulted in a significant reduction of the viability of the conidia of these three Aspergillus species as a result of lethal membrane lesions. All Aspergillus species were similarly susceptible to cupric sulphate and sodium hypochlorite. Therefore, heating, microwave and the chemical treatments tested impaired significantly the viability of Aspergillus conidia, supporting the use of these methods as preventive measures among patients at risk.
Dried monolayers of Chroococcidiopsis sp. 029, a desiccation-tolerant, endolithic cyanobacterium, were exposed to a simulated martian-surface UV and visible light flux, which may also approximate to the worst-case scenario for the Archean Earth. After 5 min, there was a 99% loss of cell viability, and there were no survivors after 30 min. However, this survival was approximately 10 times higher than that previously reported for Bacillus subtilis. We show that under 1 mm of rock, Chroococcidiopsis sp. could survive (and potentially grow) under the high martian UV flux if water and nutrient requirements for growth were met. In isolated cells, phycobilisomes and esterases remained intact hours after viability was lost. Esterase activity was reduced by 99% after a 1-h exposure, while 99% loss of autofluorescence required a 4-h exposure. However, cell morphology was not changed, and DNA was still detectable by 4',6-diamidino-2-phenylindole staining after an 8-h exposure (equivalent to approximately 1 day on Mars at the equator). Under 1 mm of simulant martian soil or gneiss, the effect of UV radiation could not be detected on esterase activity or autofluorescence after 4 h. These results show that under the intense martian UV flux the morphological signatures of life can persist even after viability, enzymatic activity, and pigmentation have been destroyed. Finally, the global dispersal of viable, isolated cells of even this desiccation-tolerant, ionizing-radiation-resistant microorganism on Mars is unlikely as they are killed quickly by unattenuated UV radiation when in a desiccated state. These findings have implications for the survival of diverse microbial contaminants dispersed during the course of human exploratory class missions on the surface of Mars.
OBJECTIVES: Apigenin is a potent inhibitor of glucosyltransferases and tt-farnesol affects the membrane integrity of Streptococcus mutans. We investigated the influence of apigenin and tt-farnesol, alone and in combination, on the accumulation, polysaccharide composition and viability of S. mutans UA159 biofilms. METHODS: Initially, biofilms were grown for 54 h; then, the early-formed biofilms were treated for 1 min twice daily with one of the following: (i). 1.33 mM tt-farnesol; (ii). 1.33 mM apigenin; (iii). apigenin + tt-farnesol (1.33 mM each); (iv). vehicle control (20% ethanol with 0.75% dimethyl sulphoxide); (v). 0.12% chlorhexidine (1.33 mM); or (vi). physiological saline (145 mM NaCl). The procedure was repeated at biofilm ages of 78 and 102 h, and biofilms were harvested at 126 h. The dry weight, protein concentration, number of cfu, and polysaccharide composition per biofilm were determined. RESULTS: The dry weights of the biofilms treated with the test agents were significantly less (30-50%) than those treated with vehicle control (P < 0.05). Biofilms treated with the test agents also resulted in lower amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides and, to a lesser extent, fructans. The fructosyltransferase activity was affected only by apigenin and apigenin + tt-farnesol. The recoverable viable counts of S. mutans were slightly lower (0.5 to 1 log10 decrease in cfu/biofilm) after apigenin and tt-farnesol treatments compared with the vehicle control. Chlorhexidine displayed potent bactericidal activity, and virtually halted the further accumulation of early-formed (54 h old) biofilms. CONCLUSIONS: Apigenin and tt-farnesol affected the accumulation and polysaccharide content of S. mutans biofilms without major impact on the bacterial viability.
Neisseria meningitidis SD1C exhibited a low tolerance to penicillin G (0.03 microgram/ml). Loss of viability in the absence of polyvinylpyrrolidone-40 and horse serum was independent of the concentration of antibiotic above the minimum inhibitory concentration, whereas the rate of bacteriolysis was concentration dependent. Penicillin-induced lysis was a secondary event in this organism. At low levels of penicillin G, growth characteristics, i.e., absorbancy changes, respiratory rate, and uptake of Mg2+, appeared normal during the first 90 min in penicillin; however, viability dropped dramatically. Additionally, total cell numbers remained constant while cell mass continued to increase at a rate normal for the population. The increase in cellular mass in the absence of cell division could be observed microscopically. Only one ultrastructural change induced by penicillin correlated with the loss in viability: the loss in continuity of the outer membrane with the peptidoglycan but only at the site of septum formation. This lesion did not occur when cells were grown in media supplemented with the protective agents polyvinylpyrrolidone-40 and horse serum. Under these conditions of growth and with relatively high levels of penicillin, constant viability was maintained, but cell division no longer occurred. Cell populations treated with penicillin in the presence of the protective agents became increasingly more dependent on the presence of these agents for total viability even in the absence of penicillin in the culture.
The general stress response of the bacterium Bacillus subtilis is controlled by the sigmaB transcription factor. Here we show that loss of sigmaB reduces stationary-phase viability 10-fold in either alkaline or acidic media and reduces cell yield in media containing ethanol. We further show that loss of the developmental transcription factor sigmaH also has a marked effect on stationary-phase viability under these conditions and that this effect is independent from the simple loss of sporulation ability.
Standard reference strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were suspended in sterile deionized water and tested daily via disk agar diffusion for their antimicrobial susceptibilities. Inocula of E. coli and P. aeruginosa yielded acceptable results for up to 32 days; however, results on S. aureus were unacceptable due to loss of viability of the organism in water. E. coli and P. aeruginosa inocula, in water, can be used daily for quality control of the disk gear diffusion test.
Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.
The application of the collagenase portal vein perfusion technique for the isolation of intestinal cells resulted in the preparation of highly viable enterocytes. Cell viability was found to be greater than 90% as tested by LDH release and Trypan blue exclusion techniques. According to the results of marker enzyme determinations, collected cells were mostly of matured villus type, characterized by high disaccharidase and very low thymidine kinase activity. In vitro treatment of the isolated cells with the anticancer agent cis-diamminedichloroplatinum (II) caused decrease of the metabolic processes, i.e. glucose oxidation and protein synthesis, demonstrating that beyond the production of DNA-crosslinks other mechanisms may play a role in the cytotoxic effect of the drug. It should be stressed, however, that prolonged incubation of the cell suspension over 30 min at physiological temperature may itself lead to gradual decrease of the viability and to disturbance of the metabolic activity of the cells.
The decisive role of biotic factors in the preservation of the viability of Y. pseudotuberculosis is shown. The results of experiments suggest that protozoa may serve as reservoir in the spread of Y. pseudotuberculosis infection.
BACKGROUND: The unique, black, hypersaline mud mined from the Dead Sea shores is extensively used in mud packs, masks, and topical body and facial treatments in spas surrounding the lake, and in cosmetic preparations marketed worldwide, but little is known about its antimicrobiological properties. METHODS: We performed detailed microbial and chemical analysis of Dead Sea mineral mud compounded in dermatological and cosmetic preparations. RESULTS: Using conventional bacteriological media (with or without salt augmentation), we found surprisingly low numbers of colony-forming microorganisms in the mud. The highest counts (up to 20,000 colonies per gram, mostly consisting of endospore-forming bacteria) were obtained on sheep blood agar. Test microorganisms (i.e. Escherichia coli, Staphylococcus aureus, Propionibacterium acnes, Candida albicans) rapidly lost their viability when added to the mud. Zones of growth inhibition were observed around discs of Dead Sea mud placed on agar plates inoculated with Candida or with Propionibacterium, but not with Staphylococcus or Escherichia. The effect was also found when the mud was sterilized by gamma irradiation. Using (35)S-labeled sulfate as a tracer, bacterial dissimilatory sulfate reduction could be demonstrated at a low rate (0.13 +/- 0.03 nmol/cm(3).d). CONCLUSION: The antibacterial properties of Dead Sea mud are probably owing to chemical and/or physical phenomena. Possible modes of antimicrobial action of the mud in relation to its therapeutic properties are discussed.
Juvenoids are biologically active compounds, of relatively low toxicity to humans, that efficiently inhibit the fertility of insects. However, little attention has been paid to the stability and toxicity of products that may be generated by their biodegradation in the ecosystem. This study describes a simple comparison of the toxicity of the active compound and its degradation products generated by aerobic soil microbial isolates. Surprisingly we have found that toxicity of a biologically active carbamate juvenoid N-[2-[4-(2,2-ethylenedioxy-1-cyclohexylmethyl)-phenoxylethyl]carbamate (W328) was comparable with that of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The toxic effect was evaluated using the determination of the ATP/ADP content and viability of HeLa S3 cells exposed to various concentrations of the chemicals tested for various durations. DDT was used as a reference compound. Its toxicity was compared with two juvenile hormone analogs. The original compound, W328, was found to be the most toxic. The major product (W329) generated both by yeast isolates and the mixture of moulds lost its activity on reproduction of the tested insect. Its toxicity towards human cells was also decreased. Another two W328 degradation HPLC fractions exhibited significantly reduced toxicity compared to W328.
For oil-based products, FDA recommends substitution of the oil with a compound which has similar viscosity and physical characteristics. In this study, a substitute for oil-based products was screened by measuring the viscosity and filterability, and examined for the presence of cell clumps in the various test fluids using an optical microscopy. The viscosity of the test fluids measured in the range of about 60-75 cP. Brevundimonas diminuta (formerly Pseudomonas diminuta), a standard challenge test organism for validation of 0.2 micron rated membrane filters, formed clumps in oils (corn, olive, sesame, and soybean) and polyethylene glycol (PEG, Molecular Weight (MW) = 400 and 1,000). During the viability test, cells suspended in 80% glycerol showed a ten-fold mortality rate after an exposure for 6 hours, but there was no significant change in viability in alginate (low, medium, and high viscosity) for 24 hours. These results suggested that alginate is better suited as a substitute for oil-based products than 80% glycerol. Since high viscosity fluids take longer to filter, the glycerol mortality rate would influence the challenge test negatively. A scaled-down filtration system has been developed for the described trials, and the bacterial challenge and bubble point tests have been performed in 1.6% alginate (66.7 cP), which was the choice of carrier fluid.