Adsorption of Dissolved Organic Matter to the Inorganic Filter Substrate and Its Implications for 14C Uptake Measurements

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.     

Monitoring of the toxic dinoflagellate Karenia brevis using gyroxanthin-based detection methods

The threat to human health and fisheries resources due to blooms of the toxic dinoflagellate Karenia brevis has lead to widespread public concern and calls for continuous monitoring of coastal waters for this organism. Here, a rapid and sensitive photopigment-based monitoring approach is described that incorporates refinements to standard filtration and analytical methods. This method uses the biomarker pigment gyroxanthin-diester contained in cells of some gymnodiniod species including K. brevis. Investigations of the retention efficiencies of five filter types for gyroxanthin from natural blooms of K. brevis showed no significant differences between GF/F, GF/C, 934-AH, GF/A or GF/D filters. Retention efficiencies were generally greater than 98% of cells added, indicating that the larger nominal pore size filters may be used safely for sample collection, reducing overall filtration times for large volumes of water. Simulated bloom experiments using cultures of K. brevis added to unfiltered water from Galveston Bay showed that retention of gyroxanthin on GF/D filters was significantly lower than on other filter types. There were significant interactions (p < 0.01) between filter type and cell density for the variables gyroxanthin, gyroxanthin chl a^–1 and gyroxanthin cell^–1, suggesting that the performance of the different filter types was dependent on cell density. Retention efficiencies for the simulated blooms ranged between >99% of cells retained and <30% of cells retained (greatest losses were for the GF/D filters). Combined results of natural and simulated blooms indicated that GF/C, 934-AH or GF/A filters gave the best retention efficiency with the fastest filtration times. Sample processing times were also improved by modifying the flow gradients in an existing HPLC protocol allowing the analysis of 106 samples in 24 h. The resulting protocol is suitable for incorporation into routine water quality monitoring programs, and would greatly facilitate the early detection and tracking of K. brevis blooms in coastal waters.     

样品制备过程对测定水中溶解态微囊藻毒素的影响

系统比较了5种不同材质滤膜对于制备溶解态微囊藻毒素的影响,发现了影响藻毒素测定样品前处理的关键操作步骤。结果表明,醋酸纤维滤膜（CA filter）最多可吸附样品中79%的藻毒素,导致测得的MCs浓度严重偏低。玻璃纤维（GF/C）滤膜和聚醚砜（PES）材质滤器对制备溶解态微囊藻毒素过程影响很小。另外,发现离心法无法完全去除野外水华样品中藻细胞,反而可能导致藻细胞破裂,释放藻毒素,影响水中溶解态微囊藻毒素的测定。研究结果将对发展水体中溶解态藻毒素测定标准方法提供依据。       

Comparison of different filter types on chlorophyll-a retention and nutrient measurements

In studies on the biochemical compounds in phytoplankton, water samples generally are (pre-) filtered to retain the organisms for extraction. Such filters can be used for further investigations in microscopic or chromatographic (for example High-Performance-Liquid-Chromatography, HPLC) methods, while the filtrates can be used for nutrient or fluorometric measurements as well as for microscopic examinations. Which filter is chosen for a study often depends on its pore size, the costs and, in particular for HPLC measurements, on its chemical compatibility. In our study we compared the chlorophyll-a retention on the filters by HPLC as well as the fluorescence before and after filtration, and nutrient content of the filtrates. The filters we tested were of different material and with various pore sizes. Although Whatman GF/C and GF/F filters are preferred in phytoplankton studies, we found that the Nylon Membrane filter of 0.2 μm pore size provided the most consistent results in chlorophyll-a retention and the one of 0.45 μm pore size in nutrient investigations.     

Carbon and Nitrogen Content of Natural Planktonic Bacteria

A method of estimating carbon and nitrogen content per unit of natural bacterial cell volume was developed. This method is based on the difference in the retentiveness of bacteria between two kinds of glass fiber filter, GF/C and GF/F (Whatman, Inc., Clifton, N.J.). Biovolume and biomass (carbon and nitrogen content) of bacteria which passed through the GF/C but not the GF/F filter were estimated with an epifluorescence microscopy and a CHN analyzer, respectively. From seasonal determinations of natural planktonic bacteria in epilimnetic waters of a mesotrophic lake, the conversion factors of 106 fg of C/μm³ and 25 fg of N/μm³ were derived as average values. By using these values, the contribution of bacteria to the biomass of lake plankton is discussed.     

Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

An easy assay for histone acetyltransferase activity using a PhosphorImager

A simple radiometric assay for histone acetyltransferase (HAT) activity employing a PhosphorImager is described. In the proposed procedure, following incubation of [1-¹⁴C]acetyl coenzyme A (CoA), histones, and HAT enzyme, radiolabeled histones are fixed on GF/F glass microfiber filter while the excess of acetyl CoA is washed out. Afterward, the filter is exposed to a phosphor-screen and the resulting spot signals are quantified with a PhosphorImager. Given the small volumes required, the new assay reduces reagent consumption and contaminated waste. Moreover, the assay can be performed with a large number of samples simultaneously, is applicable on different protein substrates, and is adaptable to the analysis of other protein modifications.     

Use of the 0.22 {micro}m Millipore membrane for light-transmission measurements of aquatic particles

The use of the 0.22 µm Millipore cellulose membrane forthe measurement of the in vivo absorption of aquatic particlesby the light-transmission method has been explored relativeto the performance of the currently used 0.7 µm WhatmanGF/F glass-fiber filter. The standard solvent extraction processfor pigment removal has been replaced by a newly developed processbased on NaClO bleaching. The tests carried out indicate thatthe two filters exhibit comparable performances. It has beenshown that a double measurement, performed first in the particlesretained in the GF/F filter and then on the sample obtainedpassing the GF/F filtrate through a Millipore membrane, allowsfor the selective determination of the absorption of the particlefraction with diameter in the 0.22–0.7 µm range.     

A method for the experimental determination of light absorption by aquatic heterotrophic bacteria

A method has been set up for the experimental determinationof the volume coefficient for light absorption in vivo by aquaticheterotrophic bacteria. The application described here is theabsorption measurement of the bacterial fraction that passesthrough the commonly used GF/F ifiter and remains unaccountedfor. The experimental samples were prepared by successive waterfiltra tions through GF/F and 0.22 µm Millipore membranes.Light-transmission and light-reflection measurements of thefilter-retained samples were performed using a dual-beam spectrophotometerequipped with an integrating sphere attachment. Sample absorptionwas derived from the data by a procedure that corrects for thecontamination of the results due to the high degree of lightscattering by the bacteria. The bacterial absorption was discriminatedfrom fine detritus absorption by bleaching the bacterial respiratorypigments using a K₂S₂O₈ solution. The absorption amplificationcaused by multiple scattering in the filter was corrected forby an expression that was obtained experimentally. A test ofthe method, including error analysis, was performed on samplescollected in both marine and inland waters. The relative contributionsto light absorption by heterotrophic bacteria and various typesof particulate matter were also measured for a typical situation.Combining the measured volume absorption coefficients with backscatteringcoefficients computed by Mie theory yields a set of input datato multicomponent optical models that is needed to assess thecontribution of these heterotrophic bacteria to the radiativetransfer process.     

Conjugative transfer of the transposon Tn919 to lactic acid bacteria

Abstract The streptococcal transposon Tn 919 was transferred from Streptococcus faecalis GF590 to selected Group N Streptococcus strains and to one strain each of Lactobacillus plantarum and Leuconostoc cremoris , using the filter mating method. An S. lactis MG1363 Rif^r Tc^r transconjugant also acted as a donor, but was less efficient than GF590. Frequencies of transfer varied between 4.0 × 10⁻⁸ and 5.29 × 10⁻⁵ per recipient. Further analysis of S. lactis MG1363 Sm^r Tc^r transconjugants showed that insertion of Tn 919 into the chromosome was site-specific.     

Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates

A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.     

Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses,adenoviruses and male‐specific coliphages from seawater

Aim: To evaluate the electropositive, alumina nanofibre (NanoCeram) cartridge filter as a primary concentration method for recovering adenovirus, norovirus and male‐specific coliphages from natural seawater. Methods and Results: Viruses were concentrated from 40 l of natural seawater using a NanoCeram cartridge filter and eluted from the filter either by soaking the filter in eluent or by recirculating the eluent continuously through the filter using a peristaltic pump. The elution solution consisted of 3% beef extract and 0·1 mol l^?1 of glycine. The method using a peristaltic pump was more effective in removing the viruses from the filter. High recoveries of norovirus and male‐specific coliphages (>96%) but not adenovirus (<3%) were observed from seawater. High adsorption to the filter was observed for adenovirus and male‐specific coliphages (>98%). The adsorption and recovery of adenovirus and male‐specific coliphages were also determined for fresh finished water and source water. Conclusion: The NanoCeram cartridge filter was an effective primary concentration method for the concentration of norovirus and male‐specific coliphages from natural seawater, but not for adenovirus, in spite of the high adsorption of adenovirus to the filter. Significance and Impact of the Study: This study demonstrates that NanoCeram cartridge filter is an effective primary method for concentrating noroviruses and male‐specific coliphages from seawater, thereby simplifying collection and processing of water samples for virus recovery.     

The affinity of [3H]quinuclidinyl benzilate and some other radioligands for glass microfiber and cellulose filters in some common liquid scintillation solvents

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.     

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.     

Rapid Screening of Antibody Aggregates and Protein Impurities Using Short Gel Filtration Columns

Gel filtration (GF) is an excellent tool to acquire information about sizing (identity), purity, and the multimeric state of a protein of interest. Superdex™ 200 and Superdex 75 are outstanding GF media for such analysis. To speed up analysis and keep sample and buffer consumption at minimum, two prepacked short GF columns have been developed, Superdex™ 200 5/150 GL and Superdex 75 5/150 GL. With lengths of only 15 cm and volume of 3 ml, these columns allow rapid analysis (6–12 min/run) with minimal sample (4–50μl) and buffer consumption.Purification of antibodies often generates dimers and higher aggregated forms, and during optimization of purification protocols, many samples may need to be analyzed for aggregate content. Gel filtration (GF) provides reliable information about size and the purity of a protein of interest, especially when it is in a multimeric state. GF, however, is often time-consuming, and the analyses become a bottleneck. A new column format—3 ml bed vol and 15 cm long—has been developed, enabling rapid and reliable GF with low sample and buffer consumption, using Superdex™ 75 and Superdex™ 200 media for determination of size and purity status.     

Development and application of procedures for the highly sensitive quantification of cyclosarin enantiomers in hemolysed swine blood samples

The present study was initiated to develop a sensitive method for the analysis of cyclosarin (O-cyclohexyl methylphosphonofluoridate, GF) enantiomers in biological samples utilizing classical configurations of GC-MS and automated solid phase extraction. To achieve this goal, a specific procedure had to be developed to extract cyclosarin from swine blood samples thereby stabilising and minimising the racemisation/deracemisation of its enantiomers. The chiral stationary phase was GAMMA DEX (gamma cyclodextrin), on which GF and deuterated GF enantiomers were baseline-resolved. The limit of detection was 1 pg for (-)-GF with GC-EI-MS and 5 pg for (+)-GF with GC-NCI-MS. The absolute recovery of the overall procedure for sample preparation was 85%. After an intravenous infusion of a supralethal dose of GF in anaesthetised swine only (-)-GF could be quantified, (+)-GF was not detected.     

Biomass and Total Lipid Content Assessment of Microalgal Cultures Using Near and Short Wave Infrared Spectroscopy

The technique of near and short wave near-infrared spectroscopy was assessed with respect to analysis of dry matter and lipid content of microalgae with potential for biodiesel production. Microalgal culture samples were filtered through GF/C filter papers and spectral measurements of wet and oven dried (60 °C overnight) filter papers over the ranges of 300–1,100 nm and 1,100–2,500 nm were recorded. Partial least square models on culture biomass and lipid content for combined species data were poor in terms of RMSECV, R _CV and the ratio of RMSECV to SD. A single species model for C. vulgaris based on 1,100–2,500 nm spectra of dry filtrate supported a model with RMSECV, R _CV and SDR values of 0.32 g L^?1, 0.955 and 3.38 for biomass and 0.089 g L^?1, 0.874 and 2.06 with lipid, respectively. However, the dry filtrate models on biomass and lipid content performed poorly in the prediction of samples drawn from an independent series of C. vulgaris cultured under N-, P- and Fe-limited growth trial. Thus, while the near-infrared spectroscopy technique has potential for assessment of dry matter and lipid content of microalgal cultures using a dried filtrate sample, further work is required to examine the limits to model robustness.     

Detection and distribution of rotavirus in raw sewage and creeks in São Paulo, Brazil.

Rotaviruses were concentrated from 8-liter samples of raw domestic sewage and sewage-polluted creek water by adsorption to and elution from positively charged microporous filters (Zeta Plus 60S), followed by ultracentrifugation of the filter eluates. Indirect immunofluorescence and direct immunoperoxidase methods allowed detection and enumeration of rotavirus in 6 (20.6%) of 29 sewage samples and in 19 (34.5%) of 55 creek water samples. Levels of rotaviruses ranged from < 3 to 63 focus-forming units (FFU)/liter, and the geometric means were 2.2 FFU/liter in sewage, 2.9 FFU/liter at creek Tremembé, and 2.6 FFU/liter at creek Pirajussara. Wastewater samples examined during autumn and winter months showed a higher rate positivity for rotavirus than those collected in spring and summer, corresponding to the seasonal variation of rotaviral diarrhea in the city of São Paulo.     

Phenotypic characterization of tannin–protein complex degrading bacteria from faeces of goat

Tannin–protein complex degrading bacteria after enrichment were isolated from unadapted goat faecal samples. Based on the morphological, hemolytic and biochemical characters, the isolates were categorized in two groups comprising GF1–GF4 and GF5–GF6. All the isolates were gram-positive cocci, catalase negative belonging to different strains of Group D streptococci, Enterococcus faecalis. Among six isolates, GF1 was the most resistant that could tolerate up to 4% of tannic acid in the medium with no significant change in the morphology. Tannase activity was detected in all the isolates, indicating their tannin degrading potential while gallate decarboxylase activity was detected only in three isolates GF1, GF2 and GF6.