Application of the membrane filter technique to bromodeoxyuridine immunochemistry for exfoliative cytology

The membrane filter technique for smear specimens of tumors in bromodeoxyuridine (BrdU) immunochemistry is described. The staining results of Raji cells processed using the filter technique was compared with that obtained by the conventional cytospin method. Although the BrdU mean labeling index (LI) for in cytospin specimens was almost the same as the LI in membrane filter specimens, filter specimens showed excellent staining and less cell destruction compared with those processed by cytospin. Small amounts of tumor specimens such as squamous cell carcinoma and polymorphous low-grade adenocarcinoma also were processed using the membrane filter appliance. For squamous cell carcinoma, the LI for the filter specimens was 5.36 ± 0.38 and that of the paraffin sections was 5.56 ± 0.38. The membrane filter technique provided relatively undamaged specimens for exfoliative cytology and will be useful for immunohistochemical evaluation of tumor cells and for routine, noninvasive cytological screening.     

A Method for the Removal from Membrane Filters of Micro-Organisms Filtered from Water and Air

S ummary : The micro-organisms in tap water were quantitatively (99%) retained by Oxoid membrane filters. The trapped organisms were extracted from the membrane filter by a simple washing process using glass beads or a magnetic stirrer. For counts on air, the membrane filter was considerably more efficient than an ammonium alginate filter in trapping bacteria, but both techniques were equally satisfactory for moulds.     

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes.

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.     

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.     

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be applied to the detection of various chemical contaminants. Silver nanoparticles (Ag NPs) are synthesized via reduction of silver nitrate by sodium citrate. Then the NPs are aggregated by sodium chloride to form nanoclusters that could be trapped in the pores of the filter membrane. A syringe is connected to the filter holder, with a filter membrane inside. By loading the nanoclusters into the syringe and passing through the membrane, the liquid goes through the membrane but not the nanoclusters, forming a SERS-active membrane. When testing the analyte, the liquid sample is loaded into the syringe and flowed through the Ag NPs coated membrane. The analyte binds and concentrates on the Ag NPs coated membrane. Then the membrane is detached from the filter holder, air dried and measured by a Raman instrument. Here we present the study of the volume effect of Ag NPs and sample on the detection sensitivity as well as the detection of 10 ppb ferbam and 1 ppm ampicillin using the developed assay.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

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 FAILURE OF PHENOL TREATED ESCHERICHIA COLI TO GROW ON MEMBRANE FILTERS

SUMMARY: Counts of Escherichia coli were done on nutrient agar (control), on membrane filters on nutrient agar and on membrane filters on filter paper pads. With untreated bacteria counts were similar under all conditions, though membrane filters on nutrient agar tended to give slightly low counts. Phenol treated bacteria gave much lower counts when membrane filters were used: the mean counts for 3 strains of the test organism with filters on nutrient agar varied from 35–65% of the control, while counts with filters on filter paper pads were somewhat lower, varying from 30–47% of the control. The low counts on membrane filters on filter paper pads were not due to adsorption of phenol by the filters or to a low concentration of nutrients in the growth medium.     

Improvement of the membrane filter technique for enumeration of enterococci in water

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

Isolierung und Zählung von Bodenactinomyceten auf Erdplatten mit Membranfiltern

Summary A technique is described to separate actinomycetes from other micro-organisms with membrane filters on soil plates. A measured quantity of soil dilution was passed through a membrane filter and the filter with the adhering organisms was placed upside down on a medium provided with soil. After an incubation period of two weeks all actinomycetes had penetrated the filter developing colonies on the sterile surface, whereas the other organisms were retained by the filter. By this procedure the counting and isolation of actinomycetes has been facilitated. The selection of bacteriostatic active forms is practicable by stamping the crowded filter on a medium that contains test bacteria.

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Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance

Membrane fouling commonly occurs in all filter types during virus filtration in protein‐based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose‐based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post‐adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose‐based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379–386, 2018     

Filter assay technique and quench-flow experiments: examples of receptor-mediated transmembrane ion-exchange measured with membrane vesicles.

Modifications to a quench-flow apparatus are described which allow a rapid, in-line filter assay with immediate washing, in conditions to give minimum background. A design for an effluent spout is presented, which decelerates the liquid by a large factor, prevents splashes, limits the area of the filter exposed to the sample and allows an immediate wash over a larger area. A design for a filter assay funnel for general use is also presented. These devices feature minimal contact of the funnels with the filter disc. Examples are given in which in-line filtration was used to follow transmembrane ion flux in membrane vesicle preparations. In measurements of transmembrane flux with membrane vesicles and radioisotope the filter assay background can be resolved into three components. These are, (1) the uptake of radioactivity by the filter, (2) the radioactivity inside the vesicles not taking part in the specific measurement and (3) the occlusion of radioactivity in aggregated membrane particles on the filter. These different components depend on the conditions in different ways. Techniques for minimizing the background in filter assays are discussed. The importance of rapid filtration and immediate washing is demonstrated. The examples given illustrate that the function of the acetylcholine receptor from E. electricus is not affected by diisopropylfluorophosphate in the conditions used, and that added GABA is not removed from solution in a brain membrane preparation by the GABA uptake mechanisms in the short times of the experiments.     

CFD-aided design of a dynamic filter for mammalian cell separation

In the present work, a rotating disk filter was designed for mammalian cell separation with the aim of avoiding both cell damage and membrane fouling. Different geometric and operational variables of the rotating disk filter were studied using computational fluid dynamics (CFD) by varying rotor radius, rotor angle, membrane-rotor distance, and angular velocity. The combinations of these variables followed a statistical design, so that an analysis of the CFD results provided correlations describing the average shear stress on the membrane surface and the maximum shear stress in the whole module as a function of the variables studied. Based on these correlations, and on the shear resistance levels of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cell lines, which were investigated using a cone-and-plate viscosimeter, it was possible to determine the geometry and angular velocity that would minimize both cell damage and membrane fouling. After construction, the filter was tested in filtration experiments at increasing permeate fluxes. Cell viability remained >90% for the duration of the experiments (2.5 h), and no indication of fouling was observed. It was shown that the designed dynamic filter is able to effectively avoid both cell damage and membrane fouling, and thus can be used for mammalian cell harvesting and perfusion.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Studies of electric capacitance of membranes

Summary The electric capacitance and conductance of a model membrane composed of a hydrophobic filter paper and a synthetic lipid analogue, i.e., dioleylphosphate, immersed in an electrolyte solution were observed with various frequencies ranging from 20 to 3×10⁶ Hz. With successive increase of salt concentration in the external solution, the capacitance and conductance of the membrane increased discontinuously at a certain critical value of the external salt concentration. This variation of the capacitance and conductance of the membrane with the salt concentration was found to be reversible, and the critical value of salt concentration was independent of the adsorbed quantity of the lipid, and of the pore size of the filter paper as far as the adsorbed quantity of the dioleylphosphate was large.A theoretical analysis based on the membrane model for the filter paper-phospholipid system proposed in Part I of this series revealed that the dioleylphosphate impregnated in the filter paper changed its conformation from oil droplets or globular micelles to a number of bilayer membranes when the salt concentration reached the critical value for a given pair of electrolyte species and the membrane. The conformational change of the lipid analogue in the filter paper is discussed in connection with the ability of formation and stability of a black bilayer membrane of the dioleylphosphate.     

A filter assay for binding of labeled ligands to membrane-bound receptors

A filter assay was developed for the specific binding of labeled ligands to the acetylcholine receptor in an unpurified suspension of membrane fragments from Torpedo californica. Binding to the filter membrane and the standard deviation of replicates were studied, and it was possible to reduce relative standard deviation to about 3% by accounting for the variable weight of the particles dispensed, stopping the loss of particles to the filter apparatus, regulating the transmembrane pressure during filtration, controlling the time the filter remains subject to suction after all the liquid has passed through the filter, and correcting for variable counting efficiency. A method for dispensing equal aliquots of suspended particulates is also described.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.     

Modification of delayed-incubation procedure for detection of fecal coliforms in water.

Three holding media, including the vitamin-free Casitone holding medium (m-VFC) recommended by Standard Methods for the Examination of Water and Wastewater for use with the delayed-incubation membrane filter procedure, were compared for their ability to maintain viability of fecal coliforms. Each medium was tested according to the procedure described in the above reference with 60 to 80 pure cultures of fecal coliforms and a variety of natural water samples containing fecal coliforms. Fecal coliform recovery with m-ST holding medium (containing ethanol, sulfanilamide, and Tris [pH 8.6] was significantly greater than recovery with m-VFC (containing vitamin-free casein hydrolysate, sodium benzoate, sulfanilamide, and ethanol). Recovery with m-VFC, was, in turn, significantly greater than with NSB medium (containing nutrient broth, boric acid, and NaCl as major ingredients). Fecal coliform counts obtained with m-ST by the delayed-incubation membrane filter procedure were higher than counts obtained by the standard immediate incubation. This result suggested that some of the sublethally injured fecal coliforms in natural water samples may have recovered during the incubation period. We propose that m-ST be used in place of m-VFC for the delayed-incubation membrane filter procedure.