Infrared color photomicrography of soil microorganisms.

The infrared color photomicrography technique for detecting unstained microbial cells in soil is revised for use with Kodak's recently introduced Ektachrome infrared film. Thes new version of the film required achromatic objectives instead of apochromats, and the use of a blue filter in addition to the previously required red filter. These filters also improve the visual focus capability of the operator. It was found that dry soil smears can be photographed as either immersion oil or aqueous mounts. Several makes of bright-field transmitted-light microscopes gave similar results. Phase microscopy, however, produced deteriorated image quality although the proper 'false' colors were produced.     

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.     

THE USE OF MILLIPORE FILTERS IN ULTRASTRUCTURAL STUDIES OF CELL CULTURES AND VIRUSES

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 x 10⁸ virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.     

Development and application of new positively charged filters for recovery of bacteriophages from water

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.     

Development and application of new positively charged filters for recovery of bacteriophages from water.

Electronegative and electropositive filters were compared for the recovery of indigenous bacteriophages from water samples, using the VIRADEL technique. Fiber glass and diatomaceous earth filters displayed low adsorption and recovery, but an important increase of the adsorption percentage was observed when the filters were treated with cationic polymers (about 99% adsorption). A new methodology of virus elution was developed in this study, consisting of the slow passage of the eluent through the filter, thus increasing the contact time between eluent and virus adsorbed on the filters. The use of this technique allows a maximum recovery of 71.2% compared with 46.7% phage recovery obtained by the standard elution procedure. High percentages (over 83%) of phage adsorption were obtained with different filters from 1-liter aliquots of the samples, except for Virosorb 1-MDS filters (between 1.6 and 32% phage adsorption). Phage recovery by using the slow passing of the eluent depended on the filter type, with recovery ranging between 1.6% for Virosorb 1-MDS filters treated with polyethyleneimine and 103.2% for diatomaceous earth filters treated with 0.1% Nalco.     

The detection of DNA-binding proteins by protein blotting.

A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.     

Use of membrane filters for the enumeration of autotrophic thiobacilli

A new membrane filter technique for field use was developed for the enumeration of either aerobic or anaerobic, autotrophic, sulfur-oxidizing bacteria in waters and soils. Immediately after collection, samples were filtered through sulfur-coated filters and incubated in selective media. Acidification or gas evolution was used as a growth indicator of aerobic and anaerobic thiobacilli, respectively, and related to the initial number of cells deposited on the filter.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

A simple method for electrophoretic selection and fractionation of poly(A)-containing RNA and poly(A).

A simple procedure, useful for quantitative and qualitative assays of poly(A)-containing RNA and poly(A), as well as for preparative purposes, is described. Glass-fiber filters with immobilized poly(U), a well-known technique for absorption of poly(A)-containing RNA, is combined with electrophoresis in a gel slab of agarose. In front of each of the two troughs in a gel slab, glass-fiber filters are inserted, one of which is impregnated with poly(U). Two identical RNA samples, e.g., split samples of total RNA from salivary glands of Chironomus tentans, are applied to the troughs and are moved electrophoretically across two different filters. The electrophoresis is conducted under conditions which promote the formation of duplexes between absorbed poly(U) and moving poly(A). While the passage of RNA chains across the control filter may take place essentially freely, RNA molecules that contain poly(A) hybridize with poly(U) fixed in the glass-fiber filter and become trapped there. The difference between resulting gel profiles [pattern of the total RNA minus the pattern of RNA not containing poly(A)] yields the electrophoretic distribution of poly(A)-containing RNA. In addition, poly(A)-containing RNA can be eluted from the poly(U) filter with formamide and subjected to electrophoresis without a subsequent precipitation in ethanol. No measurable quantities of ribosomal RNA or tRNA are retained on the poly(U) glass-fiber filters. The hybridization technique enables a quantitative retention of poly(A) molecules representing a wide range of chain lengths.     

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.     

Evaluation of five membrane filtration methods for recovery of Cryptosporidium and Giardia isolates from water samples

We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 +/- 15.4 per 10 liters) and cysts (n = 59 +/- 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 +/- 8, P = 0.015; cysts, 49.8 +/- 12.2, P = 0.4260), and SMF (oocysts, 16.2 +/- 2.8, P = 0.0079; cysts, 35.2 +/- 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.     

Evaluation of filters for recovery of Campylobacter jejuni from water.

Campylobacter jejuni has been incriminated in several large waterborne outbreaks, but it has rarely been isolated from water itself. Better methodology is needed for the isolation of C. jejuni from water. We evaluated three types of 0.45-micron microporous filters and three different pore sizes of positively charged depth filters for their ability to recover C. jejuni from seeded, sterile tap and surface water. The microporous filters tested were Millipore HA, Gelman GN6, and Zetapor. Three pore sizes of Zeta Plus depth filters (05S, 30S, and 50S) were evaluated by using an adsorption-elution technique. The overall percent recovery in both tap and surface water by microporous filters was: Zetapor, 66%; Millipore HA, 33%; and Gelman GN6, 33%. Adsorption-elution with Zeta Plus 50S allowed 89% recovery of C. jejuni. These data suggest that both the positively charged Zetapor microporous filter and the Zeta Plus 50S depth filter are effective filters for the recovery of C. jejuni from water.     

Fermentation process air filtration via cartridge filters

The use of cartridge air filters for process air sterilization in commercial fermentation has proliferated in recent years. Sterile air cartridge filter performance is discussed. The use of dispersed oil particle (DOP) tests for in situ integrity testing is described and discussed.     

Evaluation of filters for recovery of Campylobacter jejuni from water

Campylobacter jejuni has been incriminated in several large waterborne outbreaks, but it has rarely been isolated from water itself. Better methodology is needed for the isolation of C. jejuni from water. We evaluated three types of 0.45-micron microporous filters and three different pore sizes of positively charged depth filters for their ability to recover C. jejuni from seeded, sterile tap and surface water. The microporous filters tested were Millipore HA, Gelman GN6, and Zetapor. Three pore sizes of Zeta Plus depth filters (05S, 30S, and 50S) were evaluated by using an adsorption-elution technique. The overall percent recovery in both tap and surface water by microporous filters was: Zetapor, 66%; Millipore HA, 33%; and Gelman GN6, 33%. Adsorption-elution with Zeta Plus 50S allowed 89% recovery of C. jejuni. These data suggest that both the positively charged Zetapor microporous filter and the Zeta Plus 50S depth filter are effective filters for the recovery of C. jejuni from water.     

Effects of humic materials on virus recovery from water.

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

A rapid technique for the analytical and preparative isolation of transfer RNA from reaction mixtures

The isolation of enzymically modified tRNA's from enzyme reaction mixtures is usually achieved by one of two general methods. Amino-acyl synthetase reactions are easily assayed by filter paper techniques (1) in which the tRNA is precipitated on filters of materials such as cellulose ester or glass fiber. After drying, the filters can be counted by several techniques. The limitations of this technique include unknown counting efficiency and loss of the sample. In cases in which recovery of the tRNA is desired the usual procedure involves phenol extraction of the tRNA followed by selective precipitation with salts and/or organic solvents.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration.

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

Numerical analysis of the hemodynamics and embolus capture of a greenfield vena cava filter

BACKGROUND: Vena Cava filters are used to prevent pulmonary embolism in patients with deep vein thrombosis who are unresponsive to anticoagulation therapy. Various filter designs exist in the market with different characteristics distinguishing them. An understanding of the characteristics of these filters is desirable in order to develop better designs. METHODS: A computational fluid dynamical study of the flow over an unoccluded stainless steel Greenfield Vena Cava filter (Boston Scientific, Watertown, MA) to determine its properties has been performed. Simulation of flow over a filter placed axisymmetrically in a rounded inferior vena cava has been performed at a Reynolds numbers of 1000 and the consequences of the flow (by studying parameters like shear stress and stagnation zones) have been discussed. Furthermore, a new finite element based numerical method has been developed that allows the study of capturing properties of Inferior Vena Cava filters. The key idea is the introduction of a thin-wire-model (TWM) that enables a drastic reduction in the computational cost while still maintaining control on the physics of the problem. This numerical technique has been applied to evaluate the embolus capture characteristic of a Greenfield filter. RESULTS: The flow around the unoccluded filter is found to be steady and laminar at the conditions studied. A recirculation/stagnation zone develops immediately downstream of the filter head. This zone is significantly larger when the central hole is occluded. The shear stress and stagnation zone properties for such a flow over a Greenfield filter are compared with existing literature (in vitro studies). A graph showing the regions wherein clots escape or get captured has been determined by a means of numerical simulations. The data has further been analyzed to determine the probability of clot capture as function of the clot size. CONCLUSIONS: The stagnation zone formed behind the head of the Greenfield filter is found to be smaller in size when compared to that of the same filter with the central hole occluded. A map of the shear stress distribution shows a small region having the potential for thrombogenesis. The non-Newtonian properties of blood are not seen to cause much variation in the flow field when compared to the Newtonian model. However variation in the cava size leads to a significant change in the shear stresses. This study also establishes a novel method wherein computational means are used to determine the efficacy of clot capturing of filters. These techniques can further be used to compare the different characteristics among filters.     

Effects of humic materials on virus recovery from water

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.