Separation of organic dust from microorganism suspensions by partitioning in aqueous polymer two-phase systems

Conidia of Penicillium brevi-compactum and Aspergillus fumigatus, sporangiospores of Rhizopus rhizopodiformis, spores of Streptomyces griseus, and bacterial cells of Bacillus subtilis were partitioned in two-phase systems consisting of dextran, polyethylene glycol, substituted positively charged sulfonylpolyethylene glycol, and water. At a pH of 2.8 in the system, the microorganisms showed 60 to 90% affinity for the upper, polyethylene glycol-rich phase, except for cells of B. subtilis, which were entirely located in the lower, dextran-rich phase. This partition behavior was used to separate microorganisms in aqueous suspensions of peat, wood fuel chip, and straw samples from organic dust impurities prior to total count by acridine orange staining and epifluorescence microscopy. Only one extraction of the interphase and lower phase was needed to separate approximately 98% of the conidia of Penicillium chrysogenum from a suspension containing peat dust.     

Separation of organic dust from microorganism suspensions by partitioning in aqueous polymer two-phase systems.

Conidia of Penicillium brevi-compactum and Aspergillus fumigatus, sporangiospores of Rhizopus rhizopodiformis, spores of Streptomyces griseus, and bacterial cells of Bacillus subtilis were partitioned in two-phase systems consisting of dextran, polyethylene glycol, substituted positively charged sulfonylpolyethylene glycol, and water. At a pH of 2.8 in the system, the microorganisms showed 60 to 90% affinity for the upper, polyethylene glycol-rich phase, except for cells of B. subtilis, which were entirely located in the lower, dextran-rich phase. This partition behavior was used to separate microorganisms in aqueous suspensions of peat, wood fuel chip, and straw samples from organic dust impurities prior to total count by acridine orange staining and epifluorescence microscopy. Only one extraction of the interphase and lower phase was needed to separate approximately 98% of the conidia of Penicillium chrysogenum from a suspension containing peat dust.     

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

Separation of presumptive plasma membranes from mitochondria by partition in an aqueous polymer two-phase system

Light-induced absorbance changes (LIAC), indicating the reversible reduction of a b-type cytochrome, and with a possible connection to blue light photomorphogenesis, have been found in a presumptive plasma membrane rich centrifuge fraction from LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results technique membrane particles are separated according to differences in surface properties rather than size and density. LIAC could be separated into two fractions: one partitioning into the polyethylene glycol rich upper phase and another preferring the dextram rich lower phase. Mitochondria (cytochrome c oxidase) were recovered in the lower phase. A dual distribution of LIAC was found with all materials tested: corn coleoptiles, corn shoots, barley shoots and cauliflower inflorescences. About 80–90% of the cytochromes in the upper phase were related to LIAC, whereas only 10–15% of those in the lower phase were of this kind. The LIAC preferring the upper phase was probably bound to the plasma membrane, since plasma membrane vesicles are known to have a high partition in these phase systems. The lower phase LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results within one hour after the initial pelleting) for purification of presumptive plasma membranes, yielding a preparation which contained five times less mitochondrial contamination than the preparation obtained with sucrose gradient centrifugation (the 33/45% w/w sucrose interface fraction).     

Isolation of plasma membranes from mammary gland by two-phase polymer partitioning

An aqueous polymer two phase partition method was adapted for isolation of plasma membranes from mammary gland of lactating rats. Plasma membranes isolated by this method were enriched 23-fold in activity of the plasma membrane marker phosphodiesterase I and were depleted, relative to homogenates, in activities of enzymes which are markers for intracellular organelles and endomembranes. Yields of plasma membranes obtained by this method were 0.3 +/- 0.1 mg protein/g wet tissue weight. Plasma membranes were isolated more rapidly and yields were more consistent than those obtained with density gradient centrifugation methods which have been applied for isolation of plasma membranes from mammary gland.     

Purification of protein C from human plasma by precipitation and aqueous two-phase partitioning

Summary Precipitation with PEG and partitioning in PEG/dextran aqueous two-phase systems are applied to the purification of protein C, a human plasma protein with a key role in anti-coagulation. Tandem application of precipitation followed by partitioning yields a purification factor for protein C greater than that using either process individually. The results are discussed using the hypothesis that the mechanism of solvation of protein C by PEG is similar, while that for total plasma proteins is different, in the two processes.     

Highly selective medium for isolation of Listeria monocytogenes from food

A new selective medium (Al-Zoreky-Sandine listeria medium [ASLM]) was formulated to recover Listeria monocytogenes from food specimens; the medium completely inhibited common food microflora. Recognition of Listeria colonies is evident by black discoloration of the medium due to esculin hydrolysis without need for special illuminating equipment. The medium contains acriflavin, ceftazidime, and moxalactam as selective agents. Compared with Listeria Selective Agar, ASLM was equally effective in recovering L. monocytogenes. However, ASLM inhibited micrococci, enterococci, and gram-negative bacteria, especially a strain that mimicked L. monocytogenes on Listeria Selective Agar. The new medium was able to recover heat injured cells with only 15% less count than the nonselective medium.     

Purification of plasma membranes by aqueous two-phase affinity partitioning.

A rapid method for purifying rat liver plasma membranes of high purity and yield is described. Squashed liver was homogenized in an aqueous polyethylene glycol-dextran two-phase system. After phase separation and reextraction of the bottom phase with fresh top phase, the combined polyethylene glycol-rich top phases were affinity partitioned in the presence of borate buffer with new bottom phase containing dextran-linked wheat-germ agglutinin. Under these conditions the lectin selectively pulled plasma membranes into the dextran-rich bottom phase, while other membranes preferentially distributed in the top phase. The lectin-containing bottom phase was reextracted with fresh top phase before collecting the purified plasma membranes by centrifugation. This protocol resulted in a preparation that was 30- to 40-fold enriched compared to the homogenate in plasma membrane markers for both the apical and basolateral domains and had yields of 55-70%. The contamination by other membranes was low. The entire procedure was completed within 90 min. The method should be useful for purifying plasma membranes also from other sources.     

Purification of acid-proteases by affinity partitioning in aqueous two-phase systems

Summary An affinity polymer derivative was synthesized with the group specific acid protease inhibitor pepstatin attached to dextran (M.W. 500,0001). This derivative was used in an aqueous two-phase system with hydroxypropyldextran to purify crude solutions of chymosin and Endothia parasitica (EP) acid proteases. Chymosin was purified by a factor of 6.2 with an overall yield of 83%. EP protease was similarly purified. A new pepstatin binding protease was discovered in crude EP extracts.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.     

Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning

Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH(2)PO(4)) in aqueous solution. beta-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH(2)PO(4) were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two-phase partitioning

A method of enzyme release and aqueous two-phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze-thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two-phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG-ampicillin, PEG-aniline, PEG-phosphate, and PEG-trimethylamine, were synthesized and used in aqueous two-phase systems; PEG-trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG-trimethylamine, with 0.7M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. (c) 1992 John Wiley & Sons, Inc.     

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand

The effect of the important ATPS- and bufferparameters on IgG and hybridoma partitioning in ATPSscontaining a PEG-dye-ligand was studied. Objective wasto establish selection criteria for effective ligandsfor extractive fermentations with animal cells inATPSs.In the presence of 1% PEG-dye-ligand the binding ofIgG to the PEG-ligand was affected severely by theNa-chloride concentration. The tie-line length and pHaffected IgG partitioning to a lesser extent. Thedesired partitioning of IgG into the top phase, wasonly obtained when, in addition to the 10 mmol/kgK-phosphate buffer, no Na-chloride was present. In anATPS culture medium, with ± 35 mmol/kg Na-bicarbonateand 60 mmol/kg Na-chloride, increasing thePEG-dye-ligand concentration up to 100% did increasethe partition coefficient, but was not effective inconcentrating the IgG in the top phase of ATPS culturemedium at a pH of 7.8.Furthermore, addition of the PEG-dye-ligand to ATPSculture medium changed the hybridoma cell partitioningfrom the bottom phase to the interface.     

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.