SOME PROBLEMS INHERENT IN TRANSPORT STUDIES IN SYNAPTOSOMES

A technique utilizing a 30-place manifold has been developed to study synaptosomal transport; some problems associated with such studies have been identified and clarified. The time course of L-glutamic acid uptake has been used to test variations in experimental protocol. Synaptosomes apparently become increasingly labile with increased time of incubation. This is indicated by a drop in the curve of uptake vs time after 8–12 min. Ninety seven to 98% of the glutamate taken up from a 10^?6m solution is released by osmotic shock. Synaptosomes can be stored in 0.32 m ice-cold sucrose suspension for periods up to 50 min without decline in measured uptake. Storage for 3 h or more results in a very substantial decline in measured uptake. Neither the decline in measured uptake with time, nor the decline with storage, is prevented by increasing the osmolarity of the solutions used or by use of synaptosomes from the initial 1085 g supemate rather than after sedimentation and resuspension. Although prewarming synaptosomes at 30°C for 20 min prior to their use lessened or eliminated the decline following peak uptake, the difference between stored and non-stored synaptosomes was not improved. Uptake was also much less when synaptosomes were used from the first supernate or when warmed prior to their use. Storage of tissue prior to homogenization resulted in synaptosomes that gave minimal reductions in measured uptake. Washing synaptosomes after separation from incubation medium resulted in a variable loss of substrate radioactivity, depending on such variables as brand of filter, pore size, composition of wash solution, and temperature of wash solution. The results support the hypothesis that washing causes lysis of a portion of the synaptosomes. However, with Millipore filters (0.45 μm) and a 30°C Krebs-Henseleit wash solution, the loss caused by washing is minimized (about 15%). Measured uptake is found to depend on the type of filter used. Uptake is much greater with Millipore 0.45 pm filters than with Gelman 0.45 μm filters. Use of Nuclepore (0.4 μm) filters results in measured uptakes only about 5% of that when Millipore 0.45 μm filters are used. With Millipore filters, 0.30 μm pore size filters gave uptakes only 68% of that using 0.45 pm pore size filters.     

Preparation of stable and solvent-free model membranes.

A method for the preparation of solvent-free, phospholipid-impregnated filters is described. Polycarbonate filters of 13 mm diameter, 5 μm thickness and 0.1 μm pore size were employed, and 20 to 48 nmol phospholipid per filter was incorporated. Passive permeation of polar substances across the filter was determined by a flow-dialysis procedure. The presence of phospholipid led to a decrease in passive permeability by 96 – 99%. Due to their size and stability, and a partially preferential lipid orientation, these phospholipid-impregnated filters may serve as an alternative type of model membrane.     

Studies on the Minimum Reproducible Unit of Staphylococcal L-Forms

The minimum size of a reproducible unit of staphylococcal L-forms was determined by filtration and electron microscopic methods. Ultrathin sections of an induced strain of Staphylococcal L-forms (STA-EMT-1) in liquid medium revealed several types of structures, all of which were bound by a single membrane and most of which possessed ribosome-like granules. Many of the small granules were less than 0.3 μm and were attached to the membrane of the large bodies. Using a serial filtration method, it was observed that viable L-forms were still detected in 0.22 μm filtrate, but the viable cell count of L-forms decreased in number with the decrease in pore size of membrane filters. A fractionation technique, using L-forms filtered through a membrane filter with a 0.45 μm pore size, revealed that there were three classes of small bodies but only the first class with ribosome-like granules over approximately 0.2 μm in diameter seems to be able to reproduce.     

On the mechanisms of transfilter induction of kidney tubules.

Induction of tubule formation in metanephrogenic mesenchyme by spinal cord, occurs across 15 μm thick nucleopore filters with pores greater than 0·1 μm in diameter, but not when the pores are less than 0·05 μm. Processes from the inducer cells penetrate the 0·1 μm pores but not those of 0·05 μm diameter. These results are usually interpreted in favor of contact-mediated induction, and against diffusion-mediated induction.In this communication, we establish the feasibility of a novel and third possibility, namely thát induction is mediated by short-range diffusion of material from the inducer, and that the necessary proximity (say 10 nm) between the interactants is brought about by penetration of the filter pores by cell-processes. When the pores are less than 0·1 μm in diameter, penetration does not occur and the inducer and target are kept 15 μm apart. It is argued that this distance is too great for diffusion mechanisms to achieve a high enough concentration of the inductive message at the target surface.     

Effect of Preliminary Filtration on the Functional Characteristics of Bacterioplankton from Lake Khanka

The dependence of the functional characteristics of bacterioplankton from the loess of Lake Khanka on the pore size of filtering materials was investigated. Soluble organic matter (SOM), bacteria, and bacterial consumers adsorbed on particles suspended in the lake water were found to filter differently depending on the pore size of the filtering material. Filters with pore size 4.5 m (filters II) retained up to 20% of SOM and 20–30% of bacterial cells. Filters III with pore size 2.87 m retained almost 50% of SOM and about 40% of bacteria. The double layer of gauze no. 72 (referred to as filter I) with pores size 40 m was unable to completely retain bacterial consumers. In the case of filtrates I and II, the generation time of bacterioplankton decreased with its increasing average daily concentration. In the case of filtrate III, the generation time of bacterioplankton was minimum and did not depend on its concentration. Oxygen consumption rates per one bacterial cell and per unit biomass in filtrates increased with decreasing pore size of the filters through which they had passed. The bacterial biomass and oxygen consumption rate increased exponentially in filtrates III and logarithmically in filtrates I.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

Development of isoporous microslit silicon nitride membranes for sterile filtration applications

The widely used 0.2/0.22 µm polymer sterile filters were developed for small molecule and protein sterile filtration but are not well-suited for the production of large nonprotein biological therapeutics, resulting in significant yield loss and production cost increases. Here, we report on the development of membranes with isoporous sub-0.2 μm rectangular prism pores using silicon micromachining to produce microslit silicon nitride (MSN) membranes. The very high porosity (~33%) and ultrathin (200 nm) nature of the 0.2 µm MSN membranes results in a dramatically different structure than the traditional 0.2/0.22 µm polymer sterile filter, which yielded comparable performance properties (including gas and hydraulic permeance, maximum differential pressure tolerance, nanoparticle sieving/fouling behavior). The results from bacteria retention tests, conducted according to the guidance of regulatory agencies, demonstrated that the 0.2 µm MSN membranes can be effectively used as sterile filters. It is anticipated that the results and technologies presented in this study will find future utility in the production of non-protein biological therapeutics and in other biological and biomedical applications.     

Ultrastructure and Histochemistry of the Food Trapping Mucous Film in Benthic Filter-Feeders (Ascidians)

Transmission and scanning electron microscopy of food trapping filters or “mucous films” of several Ascidians reveal the same type of extensive net with elongate rectangular meshes. The ultrastructural appearance and mesh size varies only slightly within the group. Pore sizes from 0.2 to 0.5 μm in width and from 0.5 to 2.2 μm in length with a thickness of the filaments from 10 to 40 nm are probably representative for the nets under in vivo conditions. These observations corroborate earlier experimental results which indicate a remarkably high and constant efficiency of filtration in all species kept undisturbed. Great quantities of water are evidently filtered through these nets at relatively low resistance and particles even below bacterial size (0.5 μm) are effectively trapped. Histochemical reactions indicate the presence of both mucoproteins and acidic polysaccharide components and that the filaments are probably made up of a peptide core surrounded by polysaccharides.     

Transfllter Studies on Neural Induction in the Newt

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 μm were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Mechanisms of Cell Interaction during Primary Embryonic Induction Studied in Transfilter Experiments

The transmission mechanisms operative at different stages of neutralisation during primary embryonic induction of the newt Triturus vulgaris were studied in experiments employing Nuclepore filters placed between interactive tissue explants. The transmission time of the neuralising effect was determined with 0.2 μm Nuclepore filter. In another series of experiments the transformation of neuralised ectoderm by archenteron roof mesoderm into other parts of the CNS was studied. Although sufficiently long induction times were used no transformation into hindbrain structures could be induced across filters with pore sizes from 0.1 μm to 1.0 μm. However, electron microscopy demonstrated cytoplasmic penetration into 0.6 μm filters at 15 h of induction. The results speak against free long-range diffusion of inductive material at the stage of transformation of the neuralised ectoderm to more caudal parts of CSN and warrant a more detailed structural study of the transmission phenomenon in question.     

Optimizing techniques to capture and extract environmental DNA for detection and quantification of fish

Few studies have examined capture and extraction methods for environmental DNA (eDNA) to identify techniques optimal for detection and quantification. In this study, precipitation, centrifugation and filtration eDNA capture methods and six commercially available DNA extraction kits were evaluated for their ability to detect and quantify common carp (Cyprinus carpio) mitochondrial DNA using quantitative PCR in a series of laboratory experiments. Filtration methods yielded the most carp eDNA, and a glass fibre (GF) filter performed better than a similar pore size polycarbonate (PC) filter. Smaller pore sized filters had higher regression slopes of biomass to eDNA, indicating that they were potentially more sensitive to changes in biomass. Comparison of DNA extraction kits showed that the MP Biomedicals FastDNA SPIN Kit yielded the most carp eDNA and was the most sensitive for detection purposes, despite minor inhibition. The MoBio PowerSoil DNA Isolation Kit had the lowest coefficient of variation in extraction efficiency between lake and well water and had no detectable inhibition, making it most suitable for comparisons across aquatic environments. Of the methods tested, we recommend using a 1.5 μm GF filter, followed by extraction with the MP Biomedicals FastDNA SPIN Kit for detection. For quantification of eDNA, filtration through a 0.2–0.6 μm pore size PC filter, followed by extraction with MoBio PowerSoil DNA Isolation Kit was optimal. These results are broadly applicable for laboratory studies on carps and potentially other cyprinids. The recommendations can also be used to inform choice of methodology for field studies.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

STUDIES ON MORPHOLOGY AND DEVELOPMENT OF OEDMEUS ASIATICUS ENTOMOPOXVIRUS PROPAGATED IN OEDALEUS INFERNALIS*

Abstract The morphological characteristics and development of Oedaleus asiaticus entomopoxvirus propagated in Oeddeus infernalis are reported. This virus mainly infected host's fat bodies and hemocytes. Three kinds of spheroids were observed during propagation: big spheroid (30. 41 μm × 25. 40 μm), ellipsoid (6. 58 μm × 4. 78 μm) and small spheroid (3. 35 μm × 2. 60 μm). The virions embeded in them were oval, measuring 230 nm × 176 nm. The typical characteristic of poxviruses as spherical units with the mulberry-like surface could be seen under transmission electron microscope. The lateral body was cylinder-shaped. The rope-like substances in the core folded back only once; therefore two spots in transverse sections were seen. The development of the virions included four stages: the appearance of viro-plasm, the formation of spherical particles, the differentiation of core and capsid. The grasshoppers only in the same genus could be infected by this virus.     

Membrane Filtration of the Reiter Treponeme

A membrane filtration technique with commercially available membrane filters (Millipore Corp.) was effective for the removal of Reiter treponemes from liquids such as fluorescent-antibody conjugates, to which the organisms are added for adsorption. Reiter treponemes from an 8-day culture were not microscopically detectable in filtrates through membranes with a pore diameter of 0.45 μm, but treponemes were demonstrated in the filtrate by cultural methods. No organisms of the 8-day culture passed through a membrane filter having a pore size of 0.22 μm, as determined by microscopy and culture. Culture data indicated that a filter with a pore size of 0.1 μm was necessary to prevent passage of treponemes from 4-day cultures. It is recommended that a membrane filter with a pore size of 0.22 μm or smaller be used for the removal of Reiter treponemes from suspensions and that the age of the culture be considered in choosing filter pore size.     

The use of copper and silver in carbon point-of-use filters for the suppression of Legionella throughput in domestic water systems

Aims: To evaluate throughput of seeded Legionella pneumophila bacteria in domestic point‐of‐use filters. Methods and Results: The filters were challenged with tap water seeded with Leg. pneumophila. After multiple challenge events (4·25 × 10¹¹ CFU per filter), the levels of Legionella were lower in the effluent from the filter containing both copper and silver (mean 4·48 × 10³ CFU ml^?1) than in the effluent from the filter containing copper only (1·26 × 10⁴ CFU ml^?1; P < 0·001). After a single challenge event of approx. 5 × 10⁹ CFU L. pneumophila per filter, there was no significant difference between the levels of Legionella in the effluents from a carbon filter containing copper and a carbon filter with no metals (mean 6·87 × 10² and 6·89 × 10² CFU ml^?1, respectively; P = 0·985). Conclusions: Legionella was detected in filter effluent up to 6 weeks after being challenged, indicating that while filters may reduce the levels during an initial contamination event, the exposure is extended as the accumulated bacteria slough off over time. Significance and Impact of the Study: This study has provided an understanding of the response of Legionella to the use of silver and copper in domestic point‐of‐use carbon filters.     

The effect of filtration method on the efficiency of environmental DNA capture and quantification via metabarcoding

Environmental DNA (eDNA) is a promising tool for rapid and noninvasive biodiversity monitoring. eDNA density is low in environmental samples, and a capture method, such as filtration, is often required to concentrate eDNA for downstream analyses. In this study, six treatments, with differing filter types and pore sizes for eDNA capture, were compared for their efficiency and accuracy to assess fish community structure with known fish abundance and biomass via eDNA metabarcoding. Our results showed that different filters (with the exception of 20‐μm large‐pore filters) were broadly consistent in their DNA capture ability. The 0.45‐μm filters performed the best in terms of total DNA yield, probability of species detection, repeatability within pond and consistency between ponds. However performance of 0.45‐μm filters was only marginally better than for 0.8‐μm filters, while filtration time was significantly longer. Given this trade‐off, the 0.8‐μm filter is the optimal pore size of membrane filter for turbid, eutrophic and high fish density ponds analysed here. The 0.45‐μm Sterivex enclosed filters performed reasonably well and are suitable in situations where on‐site filtration is required. Finally, prefilters are applied only if absolutely essential for reducing the filtration time or increasing the throughput volume of the capture filters. In summary, we found encouraging similarity in the results obtained from different filtration methods, but the optimal pore size of filter or filter type might strongly depend on the water type under study.     

Effect or preliminary filtration on the functional characteristics of bacterioplankton from Lake Khanka]

The dependence of the functional characteristics of bacterioplankton from the loess of Lake Khanka on the pore size of filtering materials was investigated. Soluble organic matter (SOM), bacteria, and bacterial consumers adsorbed on particles suspended in the lake water were found to filter differently depending on the pore size of the filtering material. Filters with pore size 4.5 microns (filters II) retained up to 20% of SOM and 20-30% of bacterial cells. Filters III with pore size 2.87 microns retained almost 50% SOM and about 40% of bacteria. The double layer of gauze no. 72 (referred to as filter I) with pores size 40 microns was unable to completely retain bacterial consumers. In the case of filtrates I and II, the generation time of bacterioplankton decreased with its increasing average daily concentration. In the case of filtrate III, the generation time of bacterioplankton was minimum and did not depend on its concentration. Oxygen consumption rates per one bacterial cell and per unit biomass in filtrates increased with decreasing pore size of the filters through which they had passed. The bacterial biomass and oxygen consumption rate increased exponentially in filtrates III and logarithmically in filtrates I.     

Interference of tooth differentiation with interposed filters

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.     

Formation of bacterial streamers during filtration in microfluidic systems

Bacterial behavior during filtration is complex and is influenced by numerous factors. The aim of this paper is to report on experiments designed to make progress in the understanding of bacterial transfer in filters and membranes. Polydimethylsiloxane (PDMS) microsystems were built to allow direct dynamic observation of bacterial transfer across different microchannel geometries mimicking filtration processes. When filtering Escherichia coli suspensions in such devices, the bacteria accumulated in the downstream zone of the filter forming long streamers undulating in the flow. Confocal microscopy and 3D reconstruction of streamers showed how the streamers are connected to the filter and how they form in the stream. Streamer development was found to be influenced by the flow configuration and the presence of connections or tortuosity between channels. Experiments showed that streamer formation was greatest in a filtration system composed of staggered arrays of squares 10 μm apart.