A rapid filtration assay for cAMP

The receptor-binding assay for cAMP was improved by using polyethylenimine-treated glass filters. A polyethylenimine-treated glass filter has high protein binding capacity. This high capacity allows an increase in the amount of protein per assay tube and the use of a crude preparation, such as a beef heart extract, as specific binding protein instead of a purified protein, which has been used in the classical filtration assays involving cellulose ester filters. Since the time required for the separation of the protein-cAMP complex and the free nucleotide can be shortened by the use of polyethylenimine-treated filters, the dissociation of the bound ligand during the separation procedure, which is a serious problem with other modified assay methods involving charcoal adsorption, is minimized. Filtration through polyethylenimine-treated glass filters also gives low blanks and prevents the loss of protein or ligand due to breakage of the filters, which is often observed with fragile cellulose ester membranes. In consequence, this simple and rapid filtration assay allows more accurate and reproducible determinations.     

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.     

Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies

We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-mum-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100 degrees C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.     

Adherence of bacteria, yeast, blood cells, and latex spheres to large-porosity membrane filters.

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Na, k, and nonspecific solute requirements for induction and function of galactose active transport in an antarctic psychrophilic marine bacterium

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Virus safety of plasma products using 20 nm instead of 15 nm filtration as virus removing step

During the manufacture of human plasma derivatives, a series of complementary measures are undertaken to prevent transmission of blood-borne viruses. Virus filtration using 15 nm (Planova15N) filters has successfully been implemented in manufacturing processes for various plasma derivatives primarily because virus filtration is a technique, mild for proteins, that can effectively remove even small non-lipid-enveloped viruses, such as HAV and parvovirus B19. However, the use of 15 nm filters has limitations with regard to protein capacity of the filters and the process flow, resulting in an expensive manufacturing step. Therefore, studies were performed to test whether the use of 20 nm (Planova20N) filters, having different characteristics compared to 15 nm filters, can be an alternative for the use of 15 nm filters.It is shown that 20 nm filtration can be an alternative for 15 nm filtration. However, the virus removal capacity of the 20 nm filters depends on the plasma product that is filtered. Therefore, an optimisation study must be performed with regard to process parameters such as pressure, pH and protein concentration for each plasma product. In this study, using optimised conditions, the virus removal capacity of 20 nm filters appears to be comparable or even better when compared to that of 15 nm filters.     

LOSS OF NUTRIENTS FROM WATER SAMPLES BY FILTRATION AND ITS AFFECT ON ALGAL BIOASSAY PROCEDURES

SUMMARY

Water samples were collected from fourteen sampling points along the Hunyani River system and subjected to various filtration treatments involving glass fibre filters and 1,2 and 0,45 pm membrane filters. Chemical analyses of the filtered waters showed that nitrogen, phosphorus and iron were lost by different filtration treatments. Filtration by membrane filters led to a reduction of algal growth potential as demonstrated by algal bioassays using Selanastrwn capricornutum Printz as the test alga.

Algal bioassays showed that biologically available phosphorus was primarily removed by the 1,2 μm membrane filter while biologically available nitrogen and iron was principally lost by filtration through the 0,25 μm membrane filter. A refined algal bioassay designed to determine the identity of limiting micronutrients found that all essential micronutrients were affected by membrane filtration.     

The prevention of an artifact in receptor binding assay by an improved technique

The intensity of vacuum suction used during filtration of cerebral membranes on glass fiber filters used in the assays of neurotransmitter receptors is a factor in determining the apparent extent of bound radioactivity. This is not related to protein loss during filtration at various speeds nor to rates of dissociation of various ligand-receptor complexes. It appears to be due to the failure of hydrophobic ligands on dry filters, to be readily removed by aqueous media. A means whereby the vacuum can be held constant is described and shown to eliminate this artifact.     

Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles

A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of ²²Na⁺ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist-induced uptake of ²²Na⁺ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists (‘desensitization’) can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles.     

Decomposition of Urea by the Larger Particulate Fraction and the Free Bacteria Fraction in a Pond Water

An aliquot of a water sample taken from an artificial pond on the campus of the Faculty of Science, Tokyo Metropolitan University, was filtered with Whatman GF/C glass fiber filters. Urea was added to the filtered and unfiltered waters, respectively to a final concentration of around 10 μg-at-N/l. These samples were kept in 10 liter glass bottles, which were kept on the surface of the pond with rope. Changes in the concentrations of urea, ammonium, nitrate, nitrite, and the number of bacteria were traced from 15 June through 13 July, 1973. The urea added to the unfiltered water decreased to about 1 μg-at-N/l within first three days. The decrement of the urea seemed to proceed as a first-order reaction with rate constant of 0.73 day⁻¹. On the other hand, the urea added in the filtered water kept nearly the initial concentration for 18 days. As the number of bacteria in the filtered and unfiltered water were not significantly different, decrease of the urea in the unfiltered water may be ascribable to the participate fraction removed by the filtration. The urea was not decomposed by free bacteria.     

Bacteriology of Air-Conditioning Ducts with Special Reference to Operating Rooms

The number of bacteria in air, before filtration with five different easily available filters in the low positive-pressure type of airconditioning system of the Winnipeg General Hospital, was between 3 and 4/cu. ft., and after filtration between 1 and 2/cu. ft. with all types of filters. Cl. welchii contributed about 1% and Staph. pyogenes about 0.1% of this total. Sampling the exhaust air from an operating room during an operation showed that the bacterial count fluctuated with the degree of activity in the room and was from two to 10 times as high as in the air delivered to the room.Atlhough every reasonable attempt should be made to diminish the bacterial count of air in hospitals, if much energy and money is to be spent it would probably be wiser to investigate sources of hospital infection other than the type of air-conditioning system described in this report.     

A mathematical model for the leukocyte filtration process

Leukocyte filters are applied clinically to remove leukocytes from blood. In order to optimize leukocyte filters, a mathematical model to describe the leukocyte filtration process was developed by modification of a general theoretical model for depth filtration. The model presented here can be used to predict the time-dependent leukocyte filtration as a function of cell-cell interaction in the filter, filter efficiency, filter capacity, filter dimensions, and leukocyte concentration in the suspension applied to the filter. The results of different leukocyte filtration experiments previously reported in the literature could be well described by the present model. (c) 1995 John Wiley & Sons, Inc.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration

We designed small-scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small-scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.     

Malachite green-INT (MINT) method for determining active bacteria in sewage.

A membrane filtration method was developed to determine the proportion of active (respiring) bacteria at various stages of sewage treatment. Samples were incubated in the presence of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) and, after fixation, passed through membrane filters. Filters were counterstained with malachite green and then were examined by bright-field microscopy. The contrast between bacteria and the filter background was greatly improved by drying and then clearing the filter before counterstaining. By this method, it was found that active bacterial fractions in raw sewage, settled sewage, and secondary effluent were 40, 29, and 58%, respectively, whereas the proportion of respiring bacteria in chlorinated secondary effluent was 6.1%. The active bacterial fraction of activated sludge was found to be 16%. The proposed method represents a significant improvement in speed and simplicity over existing methods for determining active bacteria in sewage.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Field diagnosis of urinary schistosomiasis by multiple use of nuclepore urine filters

It has been recognized recently that the standard field technique for the diagnosis of urinary schistosomiasis, urine filtration using reusable polyamide mesh filters, may give false-positive findings because filters cannot be washed adequately in all circumstances. In this study the alternative filtration method using polycarbonate membrane filters was tested, and the same problem existed. A variety of more vigorous washing procedures was field tested with the conclusion that polycarbonate filters can be washed adequately for reuse by a simple procedure that includes boiling for 5 min in tap water prior to washing with detergent.     

Efficacy of antimicrobial filter treatments on microbial colonization of air panel filters

AIMS: To assess the activity of biostatic agents on the microbial colonization of panel filters. METHODS AND RESULTS: Microfibre glass acrylic filters, both used and unused, were examined for the presence of microorganisms. Test strains were used to verify microbial colonization of filter media. Antimicrobial agents were applied to the filter media and tested for their ability to reduce microbial colonization. The integrity of the panel filters and the antimicrobial activity trends of the filter media treated with antimicrobials were verified. A filtration efficiency test was carried out on the treated filters to evaluate filtration performance. Filters treated with antimicrobials demonstrated markedly less microbial colonization (density and varieties of species), higher filtration efficiency and delayed deterioration of the filter. CONCLUSIONS: The most important results of this study are the demonstration of preservation of the integrity of the filters and the lower release of microorganisms from treated filters. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the resolution of problems concerning the microbial contamination of panel filters in the heating, ventilating and air-conditioning systems commonly used in the electronic industry, pharmaceutical industry, hospitals and other environments where the absence of contaminating particles and microorganisms is required.     

MODIFICATIONS IN FILTRATION METHODS FOR THE MEASUREMENT OF INORGANIC 14C UPTAKE BY PHOTOSYNTHESIZING ALGAE1

In the course, of routine primary productivity experiments with phytoplankton, the filtration step may introduce errors into the determination of inorganic ¹⁴C uptake into particulate and filtrate organic fractions. However, when ultrafine glass fiber filters, low filtration pressures, and fuming over HC1 were used, and corrections made for nonbiological adsorption of radioactivity on filters, no significant changes of cpm retained on filters per milliliter of filtration volume were found in tests with various algal populations. Cultures of Lauderia sp. and Gonyaulax polyedra, when photosynthesizing in 1 compartment of a 2-chambered diffusion apparatus, released similar amounts of radioactive dissolved organic compounds measured after diffusion or upon filtration. Under our conditions, only negligible amounts of dissolved organic compounds were retained by fillers. For many natural waters, therefore, these filtration techniques permit adequate accuracy for primary productivity measurements.     

Decomposition of urea by size-fractionated planktonic community in a eutrophic reservoir in Japan

Free bacterial populations were separated from an intact planktonic community in water of a eutrophic reservoir in Japan by filtration through Whatman GF/ C glass fiber filters (mean porosity 1.2 µm). Urea decomposition by the free bacterial populations and the intact planktonic community was determined in six different months.The separated free bacteria apparently did not take part in urea-decomposition in waters of the reservoir through the year: the number of free heterotrophic bacteria increased during the urea-decomposition experiments, however, the concentration of urea did not decrease. Whereas, in five cases out of six, urea was decomposed by the intact planktonic community. Probably, phytoplankton were responsible for most of the urea-decomposition. On the assumption that the decomposition of urea obeyed first-order kinetics, rate constants were calculated to be 0.00–0.63 day^–1 with a mean value of 0.21 day^–1.