Nontoxic and durable salt bridges using hydroxyethylmethacrylate hydrogels

Polyhydroxyethylmethacrylate polymers (poly-HEMA) form hydrogels that provide an excellent alternative to agar in the production of salt bridges for use in bioelectrochemical experiments. A method for the simple production of poly-HEMA salt bridges is described. The poly-HEMA bridges were compared with agar bridges of similar geometry. Whereas poly-HEMA salt bridges have a conductivity that is 20 times lower than that of agar bridges of a similar geometry, poly-HEMA bridges are capable of dissipating twice the power compared with agar bridges. The mechanical properties of the poly-HEMA bridges make them easy to manufacture, store, and sterilize. When agar bridges were compared with poly-HEMA bridges in long-term cell culture experiments, the failure rate of the agar bridges was found to be approximately 10% per week vs. a virtually nonexistent failure rate for the poly-HEMA bridges. Because poly-HEMA salt bridges are reliable, durable, and nontoxic to cells, they are a practical alternative to agar salt bridges in certain experimental designs.     

Thermal Injury and Recovery of Bacillus subtilis

Exposure of Bacillus subtilis NCTC 8236 to sublethal temperatures produced a change in the sensitivity of the organism to salt and polymyxin. After 30 min at 47 C, 90% of the population was unable to grow on a modified sulfite polymyxin sulfadiazine agar containing an added 1% NaCl, 1% glucose, and 1% asparagine. The data presented demonstrate that thermal injury results in degradation of both 16S and 23S ribonucleic acid (RNA) and in damage to the cell membrane, suggested by leakage into the heating mestruum of material absorbing at 260 nm. When the cells were placed in a recovery medium (Trypticase soy broth), complete recovery, indicated by a returned tolerance to salt and polymyxin, occurred within 2 hr. The presence of a protein inhibitor (chloramphenicol) and cell wall inhibitors (vancomycin and penicillin) during recovery had no effect, whereas the presence of an RNA inhibitor (actinomycin D) effectively inhibited recovery. Further data demonstrated that the injured cells were able to resynthesize both species of ribosomal RNA during recovery by using the fragments which resulted from the injury process. Also, precursor 16S and precursor 23S particles accumulated during recovery. The maturation of the precursor particles during recovery was not affected by the presence of chloramphenicol in the recovery medium.     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Fungal life in the extremely hypersaline water of the Dead Sea: first records.

The first report, to our knowledge, on the occurrence of filamentous fungi in the hypersaline (340 g salt l-1) Dead Sea is presented. Three species of filamentous fungi from surface water samples of the Dead Sea were isolated: Gymnascella marismortui (Ascomycota), which is described as a new species, Ulocladium chlamydosporum and Penicillium westlingii (Deuteromycota). G. marismortui and U. chlamydosporum grew on media containing up to 50% Dead Sea water. G. marismortui was found to be an obligate halophile growing optimally in the presence of 0.5-2 M NaCl or 10 30% (by volume) of Dead Sea water. Isolated cultures did not grow on agar media without salt, but grew on agar prepared with up to 50% Dead Sea water. This suggests that they may be adapted to life in the extremely stressful hypersaline Dead Sea.     

Purification and Properties of an Extracellular Agarase from Alteromonas sp. Strain C-1

A marine bacterial strain isolated from the Bay of San Vicente, Chile, was identified as Alteromonas sp. strain C-1. In the presence of agar, this strain produced high levels of an extracellular agarase. The production of agarase was repressed by glucose, with a parallel decrease in bacterial growth. The enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with an overall yield of 45%. The enzyme has a molecular weight of 52,000, is salt sensitive, and hydrolyzes agar, yielding neoagarotetraose as the main product, with an optimum pH of about 6.5.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed BALB/c3T3 cells

Clones were derived in culture from a tumor initiated by spontaneously transformed 3T3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of a mass population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice.     

Isolation of a bile salt sulfatase-producing Clostridium strain from rat intestinal microflora.

Bile acid sulfates, formed in human and rat livers, are desulfated by the intestinal microflora. In our study we first isolated from conventional rat feces an unnamed bacterium, termed strain S1, which desulfated the 5 beta-bile salt 3 alpha-sulfates in vitro and in vivo after association with gnotobiotic rats. Strain S1 also possessed 12 alpha-hydroxysteroid dehydrogenase and bile salt-deconjugating activities. The strain was a strict anaerobic, CO2-requiring, gram-negative, sporeforming rod and was designated as belonging to the genus Clostridium. Growth was scarce in culture media, unless in the presence of 0.1% taurine, a sulfur-containing amino acid. Addition of this substance raised the number of bacteria in thioglycolate and peptone yeast media from 10(4) per ml to 10(6) to 10(7) per ml and increased the colony diameter on agar medium from 0.2 mm to 0.5 to 0.9 mm. Sulfatase activity was specific for the 5 beta-bile salt sulfates, leaving the 5 alpha-bile salt sulfates unchanged. In addition, the sulfatase activity was cell bound, and its production was dependent on the composition of the culture medium, although no minimal sulfur medium was required for sulfatase activity.     

Destruction of Microbacterium lacticum, Escherichia coli and Staphylococcus aureus in milk by spray-drying. I. Selective count of surviving bacteria]

In this paper a method which allows the measure of microbial death rate during spray-drying by means of a streptomycin-resistant mutant that can be grown on a streptomycin-containing agar is described. Plate counts of Microbacterium lacticum, Escherichia coli, and Staphylococcus aureus recovered from skim milk powders were done on plate count agar in the presence and absence of streptomycin and on various selective media. The powders were produced from evaporated milk previously inoculated with those organisms. Our results showed that the proposed method allows the recovery of 78% of M. lacticum, 61% of E. coli, and 100% of S. aureus that survived spray-drying. Recoveries of surviving E. coli on violet bile agar and brilliant green bile 2% were 34% and 29% respectively. Baird-Parker and mannitol salt agar media allow the recovery of all surviving S. aureus, thus showing that S. aureus cells did not lose their ability to grow in media containig 7.5% NaCl. Our results show that physiological injury of the cells during spray-drying differs from injury due to heating only.     

Anticancer drug testing in vitro: Use of an activating system with the human tumor stem cell assay

The soft agar tumor colony assay has been adapted for measurement of cytotoxicity of drugs such as cyclophosphamide whose antitumor activity depends upon biotransformation to active metabolites. The S-9 fraction of rat liver, MgCl₂, KCl, glucose-6-phosphate and NADP in phosphate buffer was added to medium containing cells from various continuous human tumor cell lines in the presence and in the absence of drug. After incubation for 1 hour at 37°, cells were washed twice with medium, seeded into 0.3% agar, and plated onto 0.5% agar in petri dishes. Colonies were counted 7 to 21 days later under phase contrast microscopy. Incubation of cells from human lines with cyclophosphamide or heliotrine, an experimental antitumor agent, in the absence of complete activating system caused no or minimal inhibition of colony formation. Incubation of cell lines with cyclophosphamide or heliotrine in the presence of complete activating system markedly reduced colony formation. The cytotoxic effects of both drugs were NADP dependent. This simple technique extends the usefulness of the soft agar stem cell assay to drugs requiring microsomal activation.     

龙须菜在不同盐度胁迫下的琼胶积累及相关生理生化变化的研究

为了探究不同盐度对龙须菜生长、琼胶合成及相关基因表达的影响, 研究了15、25和35三种盐度条件下龙须菜的生长速率、琼胶含量、琼胶合成酶α -半乳糖苷酶(GLA)、半乳糖苷转移酶(GAT)、α -1,3-糖脂磺基转移酶(GST)、半乳糖-2,6-硫酸化酶(GAS)的基因表达。结果表明, 盐度25时龙须菜生长速率最高, 为7.17%/d, 高于低盐(15)时的6.27%/d和高盐(35)时的3.57%/d, 低盐和高盐都表现出明显的暗呼吸速率上升和光合速率下降。龙须菜培养15d后, 在低盐条件下琼胶含量为9.27%, 盐度25和高盐分别为6.91%和8.09%。培养第3天时在低盐和高盐下龙须菜中gla基因表达量和酶活都显著高于盐度25的表达量; 15d后在盐度25条件下gla基因表达量仍然最低, 但GLA酶活无显著差异。gat基因在低盐条件下先降低后升高, 在高盐条件下表达量始终最高, 为盐度25的3.03倍。在低盐条件下gst和gas基因都在短期内出现显著下降, 但15d后恢复到与正常盐度无显著差异。龙须菜半乳糖含量在高盐条件下相对盐度25显著升高, 达到其3.27倍, 而在低盐条件下各种单糖含量相对较低, 半乳糖含量极低。研究结果说明过高和过低盐度都会对龙须菜造成一定程度的胁迫, 并促进琼胶的合成, 但其促进琼胶积累的机制存在差异, α -半乳糖苷酶和半乳糖苷转移酶在其中扮演着非常重要的角色。     

Various aspects of protoplast production in Streptococcus lactis

The authors studied the effect of some factors, including the conditions of preincubation, the action of 2-mercaptoethanol, EDTA, alpha-amylase, on protoplast production in four strains of Streptococcus lactis caused by lysozyme. The strains differed in the nisin-producing activity and in the structure of the cell walls that were not affected with lysozyme without either preincubation in 2-mercaptoethanol or in a salt medium with minimal inhibitory concentrations of DL-threonine. EDTA and alpha-amylase increased the lysozyme effect. Among seven buffer systems studied the most favourable for protoplast production in S. lactis is the ammonia-citric buffer with EDTA, and the best regeneration medium is the agar salt medium to which, depending on the strain, either glucose or sucrose should be added as a stabilizer.     

Solute concentration effects on the expression of cellular heterogeneity of anchorage-independent growth among spontaneously transformed balb/c3T3 cells

Summary Clones were derived in culture from a tumor initiated by spontaneously transformed 3R3 cells and tested for their colony-forming efficiency in agar (CFEag). Incubation of petri dish cultures was done in subsaturation humidity to minimize mold contamination. There was great variation in CFEag between clones but also, under certain conditions, within clones. The most prominent condition that generated phenotypic diversity in CFEag was partial evaporation of the medium, which may occur during the protracted development of mass a population from a single cell. Evaporation was disproportionately great in 35-mm dishes and peripheral wells of multiwell plates. If the supraphysiological solute concentration resulting from evaporation was greater than 133% of normal, there was progressive suppression of cell growth in the succeeding transfer in agar or on plastic, even if isotonic medium was substituted 1 d before transfer. The effect of supraphysiological concentrations of all the solutes of the medium could be reproduced by simply increasing the NaCl concentration. Damaged cells were restored to their full growth potential after 3 d in isotonic medium. When nontransformed cells were chronically exposed to increased salt, irreversible increases in 2-deoxyglucose uptake were produced. With continued exposure of these cells to high salt, they became morphologically transformed, produced colonies in agar with high efficiency, and formed sarcomas when inoculated into nude mice. This work was supported by U.S. Public Health Service Grant CA 15744 from the National Cancer Institute and by Contract 03-79EV10277 from the Office of Environment, U.S. Department of Energy.     

The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

Identification of salt stress inducible genes that control cell envelope related functions in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

Plant growth promoting rhizobacteria such as Azospirillum brasilense are agronomically important as they are frequently used for crop inoculation. But adverse factors such as increasing soil salinity limit their survival, multiplication and phytostimulatory effect. In order to understand the role of the genes involved in the adaptation of A. brasilense Sp7 to salt stress, a mutant library (6,800 mutants) was constructed after random integration of a mini-Transposon Tn5 derivative containing a promoterless gusA and oriV. The library was screened for salt stress inducible Gus activity on minimal malate agar medium containing NaCl and 5-bromo-4-chloro-3-indolyl-β-d-glucuronide. Salt stress responsiveness of the promoters was estimated by quantifying GusA activity in the presence and absence of NaCl stress using p-nitrophenyl-β-d-glucuronide as a substrate. In 11 mutants showing high levels of gusA expression in the presence of salt-stress, the partial nucleotide sequence of the DNA region flanking the site of Tn5 insertion was determined and analysed using the NCBI-BLAST programs. Similarity searches revealed that 10 out of the 11 genes sequenced showed notable similarity with genes involved in functions related to modulation in the composition of exopolysaccharides, capsular polysaccharides, lipopolysaccharides, peptidoglycan and lipid bilayer of the cell envelope. Induction of cell envelope related genes in response to salt stress and salt sensitive phenotype of several mutants in A. brasilense indicate a prominent role of cell envelope in salt-stress adaptation.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

Laccase Production and Humic Acids Decomposition by Microscopic Soil Fungi

Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic substances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not established, and conditions of the enzyme production have been poorly studied. In the present work soil micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and foreststeppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied. Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was higher than without HA. Significant and weak extracellular laccase production in liquid medium was observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respectively. The level of laccase production by A. murorum was the same without inducers and in the presence of HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation requires further investigation.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium

A novel method is proposed for the production of long-chain polyunsaturated fatty acids (LCPUFA) by labyrinthulids. The method comprises a monoxenic culture with Psychlobacter phenylpyruvicus, using agar medium in which oil was dispersed. Soybean oil (SBO) was selected as the optimum material for an oil-dispersed agar medium. The labyrinthulids showed three-dimensional growth and an anastomosing ectoplasmic network in the SBO-dispersed agar medium. The oil plate changed from an opaque culture to a more transparent culture, due to growth of the labyrinthulids. The optimum culture conditions were 25-30 degrees C, an initial pH of 6-10 and artificial seawater with a salt concentration of 50-100%. These conditions are close to those where these strains were isolated. The maximum LCPUFA production (0.59 g/l) and dry cell weight (4.93 g/l) was obtained using strain S3-2 (isolated from Ishigaki Island) with 1.5% SBO at 14 days. This value was about 30 times more than that using glucose instead of SBO. The method proposed is promising in terms of the production of LCPUFA from reproducible oils.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.