Mechanism of Enhancement of Virus Plaques by Cationic Polymers

It has been assumed that plaque enhancement by cationic polymers is due to their binding of sulfated polysaccharides in agar. However, viruses that are enhanced by cationic polymers, diethylaminoethyl-dextran, and protamine were found not to be inhibited by polyanions in agar under the usual overlay conditions. In the case of adenovirus, enhancement by protamine seems to be due to the protamine serving as a source of arginine; enzymes released from the cultured cells digest the protamine and provide a reservoir of arginine for the cells. Other viruses (herpes and echovirus types 3, 4, 5, and 6) known to be susceptible to agar inhibitors were found to be enhanced by cationic polymers even under starch gel and methylcellulose overlays, which are free of polyanions. Since cationic polymers enhance the diffusion of virus through agar or starch gel, plaque enhancement seems to be the result of the gel becoming positively charged so that viruses can move effectively through them. The observation that starch gel and methylcellulose enhance plaque formation with viruses known to be inhibited under agar was also reinvestigated. When the consistency of the agar gel was reduced to the same viscosity of starch gel and methylcellulose overlays, the same plaque counts and sizes were observed under all three overlays.     

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics

Abstract A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10¹¹ cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10⁹ cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 μg/cm³ latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.     

Methane production from wastewaters by immobilized methanogenic bacteria

A methanogenic population was immobilized onto agar gel, polyacrylamide gel, and collagen membrane. Agar-gel-entrapped methanogenic microorganisms gave the highest activity. The optimum agar concentration was between 1.5 and 3% (w/v), and the optimum microbial content was 20 mg wet cells/g gel. The optimum conditions for methane production by immobilized whole cells were pH 7.0–7.5 and 37–45°C. The rate of methane production was initially 1.8 μmol/g gel/hr. Methane productivity was gradually increased and reached a steady state (4.5μmol/g gel/hr) after 25 days of incubation. The immobilized methanogenic microbial population continuously evolved methane over a 90 day period. No difference in methane productivity was observed after three months of storage at 5°C. Methane was also produced by immobilized whole cells under aerobic conditions. Furthermore, carbohydrates, such as glucose, in wastewater completely decomposed by immobilized whole cells.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

Formation of colonies of P-388 leukemic cells in semisolid agar

Substrain P-388/A2 adapted to cultivation of agar gel in the form of compact colonies was obtained as a result of alternating passages of cells of ascitic mouse leukemia P-388 in the primary semifluid agar culture and in the mouse abdominal cavity. The efficacy of colony formation and the size of the colonies depended on the initial density of the cell suspension. In case of introduction into the agar medium of 100 cells/ml the planting efficacy constituted 20%, and the number of cells in the colony by the 8th--10th days of cultivation reached 13 000.     

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.     

参杞合剂对人鼻咽癌细胞CNE细胞周期及凋亡的影响

目的研究参杞合剂(SQ)在体外对CNE细胞周期及凋亡的影响。方法应用MTT法观察参杞合剂对细胞的抑制作用,流式细胞术观察不同浓度参杞合剂作用不同时间后CNE细胞周期的改变,电镜结合DNA电泳分析参杞合剂诱导凋亡的作用。结果SQ对CNE细胞生长有明显抑制作用,且其作用强度呈现出对浓度和时间的依赖性。CNE细胞在SQ作用下随着时间的延长和浓度的增加,G0/G1期比率下降,S期比率升高,出现S期阻滞。0.0625 g.生药/ml的SQ作用48 h后诱导出凋亡,凋亡率随着浓度的增加、时间的延长而增加,电镜下可见典型凋亡小体。琼脂糖凝胶电泳呈现出凋亡特征性的DNA条带。结论参杞合剂可直接杀伤肿瘤细胞,其机制可能通过阻滞细胞周期S期,诱导肿瘤细胞凋亡实现的。     

Effects of seasons on the chemical structure and gel strength of Gracilaria pseudoverrucosa agar (Gracilariaceae, rhodophyta)

The seasonal effects on the chemical structure and rheological properties of Gracilaria pseudoverrucosa agar have been investigated using a sequential solvent extraction, ¹³C NMR and infrared spectroscopy, and gel strength measurements. The results showed that agar enriched in precursor to the agarobiose repeat unit were obtained from algae collected in summer. In contrast, algae collected in winter contained agar molecules richer in alkali-stable sulfate groups attributed in part to -galactose-4-sulfate. A similar total concentration of 6-O-methylated agarobiose repeat units was present in the agar from both algal samples but the distribution of the methylated disaccharide varied in the fractions. Agar fractions from the summer-collected sample had higher gel strength than those of the winter ones. Alkali treatment markedly improved the gel strength of the agar from the summer harvested seaweed. Different gel strengths were observed for the native and alkali-treated agar fractions extracted from the same algal sample and a gel strength comparable to that obtained for a commercial bacteriological grade agar was obtained from the alkali-treated 40% ethanol extract agar from the summer collected alga. The chemical and rheological variations due to seasonal changes are interpreted as reflecting the ratio of actively-growing (young) to resting (old) tissue in the alga and are proposed to represent a type of ‘secondarization’ of the algal cell-wall.     

Sporulation promoting factor in vegetative cells of Bacillus subtilis

A simple experimental system for detection of sporulation promoting factors was presented. This system showed that there was a sporulation promoting factor in the vegetative cells of Bacillus subtilis cultivated on nutrient agar for 9 hr (at stage T0). The factor was partially purified from the sonicate of vegetative cells by ethanol fractionation, gel filtration, chromatography and preparative gel electrophoresis, and it was identified as manganese-containing protein.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

Enhanced development and differentiation of protoplasts and spores of green and red seaweeds by a Pterocladia agar from New Zealand

Enhanced growth of macroalgae was observed on High Purity Agar, a product of Coast Biologicals Limited of Auckland New Zealand. Increases in rates of cell divisions and tissue differentiation occurred in green and red seaweeds. Protoplasts, spores, and vegetative thalli were all affected. The enhancement of growth and differentiation was concentration dependent, with high concentrations (> 1 %) appearing toxic. Few cells survived on 1 % and 1.5 % agar; over 8 weeks of culture these developed into plantlets six fold larger than those grown on an 0.3 % gel of the same agar, and as much as 11 fold larger than control plants grown on Difco Bacto agar at the same gel concentration. The activity was heat stable, water soluble, and largely bound to the carbohydrate matrix. The chemical nature of the active compound is under investigation.     

Effect of various methods of immobilization on the stability of a microbial biosensor for surfactants based onPseudomonas rathonis T

The operating and storage stability of a receptor element of an amperometric biosensor based on thePseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to Chromatographic paper GF/A, or by cross-linking induced by glutaric aldehyde. Incorporation of microbial cells in agar gel provides long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptor element of the biosensor to be rapidly recovered after measurements.     

Effect of various methods of immobilization on stability of a microbial biosensor based on Pseudomonas rathonis T during detection of surfactants

The operating and storage stability of a receptor element of an amperometric biosensor based on the Pseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to the chromatographic paper GF/A, or by the cross-linking induced by glutaric aldehyde. Incorporation of microbial cells in agar gel provides the long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptory element of the biosensor to be rapidly recovered after the measurements.     

Yeast protoplasts immobilized in alginate: Cell wall regeneration and reversion to cells

Yeast protoplasts may regenerate the cell wall and revert to cells if immobilized in a 2%–5% Ca-alginate gel and cultured in an osmotically stabilized medium. The method of protoplast immobilization and subsequent isolation from the gel is described in detail. The reversion yield is dependent of the actual gel concentration, gel shape (beads vs. sheets) and of a medium molarity, and it may be up to 90%. The morphology of the cell wall regeneration and morphology of reversion to the cell forms correspond to protoplast development in gelatin or agar gels.     

Some observations on immobilized hydrogen-producing bacteria: Behavior of hydrogen in gel membranes

A hydrogen-producing bacterium, Clostridium butyricum, was immobilized in polyacrylamide gel membrane, agar gel membrane, and collagen membrane. The apparatus consisted of a compartment(I or II) and a whole-cell-entrapped gel membrane which was placed in the center of the two compartments. The behavior of hydrogen through whole-cell-entrapped gel membrane was examined for improving the sensitivity of a microbial BOD sensor and the efficiency of biochemical fuel cells. The bacteria-polyacrylamide gel membrane produced a higher amount of hydrogen in compartment II than the other bacteria-entrapped gel membranes. The apparent diffusion constant decreased with increasing bacteria contents in the gel membrane. As a result, 100 mg wet cells/g gel was the maximal bacteria content in the membrane for the production of hydrogen. Twenty percent of the hydrogen evolved by the whole cells diffused to compartment II, and there was a linear relationship between the glucose concentration in a bulk solution (compartment I) and the rate of hydrogen diffused to compartment II.     

Effects of phosphate and nitrate supply on productivity,agar content and physical properties of agar ofGracilaria strain G-16S

Gracilaria strain G-16S was cultured in various phosphorus (P) supply rates with low or high nitrogen (N) supply to determine the effects of nutrient supply on its productivity, agar content and physical properties of the agar. Productivity was reduced after four weeks of growth in zero P supply as plants reached 0.07% P tissue content (critical level), with fragmentation of these plants by six weeks (0.05% P; minimum viable level). Native agar content was higher in low P and high N, or low N conditions. Agar content appeared to increase with decreasing P under high N supply. This increase was not apparent with alkali treatment prior to extraction. Agar gel strength was greatly increased by alkali treatment. The highest gel strengths were obtained under high N supply at all P supply rates except zero P, and under low N supply at 12 M P week^–1. Native agar gel strengths showed a similar pattern on a lower scale. Melting temperatures were higher in agars with higher gel strengths. Dynamic gelling temperatures were generally high for alkali-treated agar, with agar from plants grown in zero P supply showing a slightly elevated gelling temperature. Melting and gelling temperatures of native agars with the highest gel strengths were in the same range as bacteriological agar. These results show that P and N supply affects productivity, agar content and agar physical properties, but the tradeoffs between a slightly higher agar quantity under nutrient limitation and higher agar quality under nutrient-replete conditions seem to favor the latter.     

Diffusible Cytokinins in shoot apices of Dahlia variabilis

Cytokinin activity (soyabean callus assay) has been determinedin excised apical buds of Dahlia variabilis before and aftera period of 3 h with cut surfaces in contact with agar gel,in the agar gel and in xylem exudates from cut shoot stumps. Buds before diffusion gave three peaks of activity in the butanolfraction, one in the aqueous fraction, following paper chromatography.Two of the former diffused into agar gel, the third (in whichmost activity was recorded) decreased in level during the 3-hperiod but was absent from the agar diffusate. The water-solublecytokinin remained at its original level and was absent fromthe agar diffusate. The three peaks of activity in the butanolfraction were also present in xylem exudates. Ether and ethylacetate fractions contained callus-growth inhibitors which diffusedinto agar gel.     

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Production of cellulolytic enzymes by immobilized Sporotrichum thermophile.

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was considerably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.